A method of treating and preventing loss of cognition

ABSTRACT

Pharmaceutical compositions for delivery of active ingredient(s) and kit(s) useful to the invention are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of prior U.S. patentapplication Ser. No. 13/875,027, filed May 1, 2013, which is adivisional of U.S. patent application Ser. No. 12/791,174, filed Jun. 1,2010, which application claims the benefit of the priority of U.S.Provisional Application No. 61/187,549, filed Jun. 16, 2009, thecontents of which are all incorporated herein by reference. Applicantclaims priority to each of the foregoing related applications.

FIELD OF THE INVENTION

The present invention relates to a new treatment for hot flushes,vasomotor symptoms, and night sweats in women. In particular, thetreatment includes the administration of a precursor of sex steroids incombination with a selective estrogen receptor modulator (SERM) forreducing the risk of acquiring breast or endometrial cancer. Theinvention also provides kits and pharmaceutical compositions forpracticing the foregoing combination. Administration of the foregoingcombination to patients reduces or eliminates the incidence of hotflushes, vasomotor symptoms, night sweats, and sleep disturbance.Moreover, the risk of acquiring breast cancer and/or endometrial canceris believed to be reduced for patients receiving this combinationtherapy. Additional benefits such as reduction of the likelihood or riskof acquiring osteoporosis, hypercholesterolemia, hyperlipidemia,atherosclerosis, hypertension, Alzheimer's disease, loss of cognition,loss of memory, insomnia, cardiovascular diseases, insulin resistance,diabetes, and obesity (especially abdominal obesity) are also provided.

BACKGROUND Related Art

Set forth below are full citations of references discussed infra hereinusing more abbreviated citation format.

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It is known that a large number of diseases, conditions and undesirablesymptoms respond favorably to administering exogenous sex steroids, orprecursors thereof. For example, estrogens are believed to decrease therate of bone loss while androgens have been shown to build bone mass bystimulating bone formation. Hormone replacement therapy (e.g.,administration of estrogens) may be used for the treatment of menopausalsymptoms. Progestins are frequently used to counteract the endometrialproliferation and the risk of endometrial cancer induced by estrogens.Use of estrogens, androgenic compounds and/or progestins for treatment,or for prophylactic purposes, for a wide variety of symptoms anddisorders suffer from a number of weaknesses. Treatment of females withandrogenic compounds may have the undesirable side effect of causingcertain masculinising side effects. Also, administering sex steroids topatients may increase the patient's risk of acquiring certain diseases.Female breast cancer, for example, is exacerbated by estrogenicactivity.

In addition, androgenic compounds have been found to be beneficial forthe treatment of the mastalgia frequently caused by HRT (Pye et al.,1985). In fact, estrogen replacement therapy may result in severe breastpain which may lead to discontinuation of therapy.

More effective hormonal therapies and reduction of side effects and riskare needed. The combination therapies of the present invention, and thepharmaceutical compositions and kits that may be used in thosetherapies, are believed to address these needs.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of treatingor reducing the incidence or risk of acquiring. hot flushes, vasomotorsymptoms, night sweats, and sleep disturbance.

It is another object to provide methods of treating or reducing the riskof acquiring the above-indicated diseases, while minimizing the risk ofacquiring breast cancer and/or endometrial cancer, osteoporosis,cardiovascular diseases, hypercholesterolemia, hyperlipidemia,atherosclerosis, hypertension, insulin resistance, diabetes, obesity(especially abdominal obesity), and vaginal dryness.

It is another object to provide kits and pharmaceutical compositionssuitable for use in the above methods. Preferably, these products arepackaged with directions for using the contents thereof for reducing oreliminating the incidence of symptoms selected from the group consistingof hot flushes, vasomotor symptoms, and night sweats.

In one embodiment, the invention provides a method of reducing oreliminating the incidence of hot flushes, vasomotor symptoms, nightsweats, and sleep disturbance, said method comprising administering topatient in need of said elimination or reduction, a therapeuticallyeffective amount of a precursor of sex steroids or prodrug thereof inassociation with administering to said patient a therapeuticallyeffective amount of a selective estrogen receptor modulator or anantiestrogen or prodrug thereof.

It is preferred that the sex steroid precursor is selected from thegroup consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,4-androstene-3,17-dioneo, and a prodrug of any of the foregoingadditional agents.

In another embodiment the invention provides additional beneficialeffects or reduces the risk of acquiring a condition selected from thegroup consisting of osteoporosis, hypercholesterolemia, hyperlipidemia,atherosclerosis, hypertension, Alzheimer's disease, insulin resistance,diabetes, loss of muscle mass, obesity, said beneficial effects beingobtained by administering to patient in need of said beneficial effects,a therapeutically effective amount of a precursor of sex steroids orprodrug thereof in association with administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator or prodrug thereof.

In another embodiment the invention provides a pharmaceuticalcomposition comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) a therapeutically effective amount of at least one sex        steroid precursor or prodrug thereof; and    -   c) a therapeutically effective amount of at least one selective        estrogen receptor modulator or an antiestrogen or prodrug.

In another embodiment the invention provides a pill, a tablet, acapsule, a gel, a cream, an ovule, or a suppository comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) a therapeutically effective amount of at least one sex        steroid precursor or prodrug thereof; and    -   c) a therapeutically effective amount of at least one selective        estrogen receptor modulator or an antiestrogen or prodrug.

In another embodiment the invention provides a kit comprising a firstcontainer containing a pharmaceutical formulation comprising atherapeutically effective amount of at least one sex steroid precursoror a prodrug thereof; and said kit further comprising a second containercontaining a pharmaceutical formulation comprising a therapeuticallyeffective amount of at least one selective estrogen receptor modulatoror an antiestrogen or prodrug thereof.

In another embodiment, the invention pertains to a method of treating orreducing the incidence of hot flushes, vasomotor symptoms, night sweats,and sleep disturbance by increasing levels of a sex steroid precursorselected from the group consisting of dehydroepiandrosterone (DHEA),dehydroepiandrosterone-sulfate (DHEA-S), androst-5-ene-3β,17β-diol(5-diol) and 4-androstene-3,17-dione in a patient in need of saidtreatment or said reduction, and further comprising administering tosaid patient a therapeutically effective amount of a selective estrogenreceptor modulator (SERM) as part of a combination therapy.

As used herein, “Pure SERM” means that the SERM does not have anyestrogenic activity in breast and endometrial tissues at physiologicalor pharmacological concentrations.

In another embodiment, the invention provides a kit comprising a firstcontainer containing a therapeutically effective amount of at least oneprecursor of sex steroids and further comprising a second containercontaining a therapeutically effective amount of at least one selectiveestrogen receptor modulator.

In another embodiment, the invention provides, in one container, apharmaceutical composition comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) a therapeutically effective amount of at least one precursor        of sex steroids; and    -   c) a therapeutically effective amount of at least one selective        estrogen receptor modulator.

In another embodiment, the invention provides a method of reducing oreliminating the incidence of symptoms selected from the group consistingof hot flushes, vasomotor symptoms, and night sweats, said methodcomprising administering to a patient in need of said elimination orreduction, (i) a therapeutically effective amount of a sex steroidprecursor or prodrug thereof in association with (ii) a therapeuticallyeffective amount of a selective estrogen receptor modulator or anantiestrogen or prodrug of either.

In another embodiment, the invention provides a pharmaceuticalcomposition for reducing or eliminating symptoms selected from the groupconsisting of hot flushes, vasomotor symptoms, and night sweats,comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) at least one sex steroid precursor or prodrug thereof; and    -   c) at least one selective estrogen receptor modulator or an        antiestrogen or prodrug of either;        -   wherein said pharmaceutical composition is provided in            packaging that directs use of said composition for reduction            or elimination of at least one symptom selected from the            group consisting of hot flushes, vasomotor symptoms and            night sweats.

In another embodiment, the invention provides a kit for reducing oreliminating symptoms selected from the group consisting of hot flushes,vasomotor symptoms, and night sweats, comprising (i) a first containerhaving therein a at least one sex steroid precursor or a prodrugthereof; (ii) a second container having therein a at least one selectiveestrogen receptor modulator, or an antiestrogen or prodrug of either ofthe foregoing; and (iii) instructions for using the kit for thereduction or elimination of at least one symptom selected from the groupconsisting of hot flushes, vasomotor symptoms and night sweats.

As used herein, compounds administered to a patient “in associationwith” other compounds are administered sufficiently close toadministration of said other compound that a patient obtains thephysiological effects of both compounds simultaneously, even though thecompounds were not administered in close time proximity. When compoundsare administered as part of a combination therapy they are administeredin association with each other. Preferred selective estrogen receptormodulators discussed herein are preferably used in combination withpreferred sex steroid precursors dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol, or4-androstene-3,17-dione, especially dehydroepiandrosterone.

The estrogen replacement therapy is commonly used in postmenopausalwomen to prevent and treat diseases due to the menopause, namelyosteoporosis, hot flushes, vaginal dryness, coronary heart disease(Cummings 1991) but presents some undesirable effects associated withchronic estrogen administration. Particularly, the perceived increasedrisk for uterine and/or breast cancer (Judd, Meldrum et al., 1983;Colditz, Hankinson et al., 1995) generated by estrogen is the majordisadvantage of this therapy. The authors of the present invention havefound that the addition of a selective estrogen receptor modulator(SERM) to precursors of sex steroids administration suppresses theseundesirable effects.

On the other hand, SERMs alone have little or no beneficial effects onsome menopausal symptoms like hot flushes and sweats. The applicantbelieves that the addition of a precursor of sex steroids to SERMtreatment of menopausal symptoms reduces or even eliminates hot flushesand sweats. It is important to note that hot flushes and sweats are thefirst manifestations of menopause and the acceptation or non-acceptationof menopausal treatment by patients is usually dependent upon thesuccess or non-success in the reduction of hot flushes and sweats.

As used herein, a selective estrogen receptor modulator (SERM) is acompound that either directly or through its active metabolite functionsas an estrogen receptor antagonist (“antiestrogen”) in breast tissue,yet provides estrogenic or estrogen-like effect on bone tissue and onserum cholesterol levels (i.e. by reducing serum cholesterol).Non-steroidal compounds that function as estrogen receptor antagonistsin vitro or in human or rat breast tissue (especially if the compoundacts as an antiestrogen on human breast cancer cells) is likely tofunction as a SERM. Conversely, steroidal antiestrogens tend not tofunction as SERMs because they tend not to display any beneficial effecton serum cholesterol. Non-steroidal antiestrogens we have tested andfound to function as SERMs include EM-800, EM-652.HCl, Raloxifene,Tamoxifen, 4-hydroxy-Tamoxifen, Toremifene, 4-hydroxy-Toremifene,Droloxifene, LY 353 381, LY 335 563, GW-5638, Lasofoxifene, bazedoxifene(TSE 424; WAY-TSE 424; WAY 140424;1-[[4-[2-(hexahydro-1H-azepin-1-yl)ethoxy]phenyl]methyl]-2-(4-hydro-xyphenyl)-3-methyl-1H-indol-5-ol),Pipendoxifene (ERA 923;2-(4-hydroxyphenyl)-3-methyl-1-[[4-[2-(1-piperidinyl)ethoxy]phenyl]methyl]-1H-indol-5-ol),and Idoxifene, but are not limited to these compounds.

But we have found also that all SERMs do not react in the same mannerand may be divided into two subclasses: “pure SERMs” and “mixed SERMs”.Thus, some SERMs like EM-800 and EM-652.HCl do not have any estrogenicactivity in breast and endometrial tissues at physiological orpharmacological concentrations and have hypocholesterolemic andhypotriglyceridemic effects in the rat. These SERMS may be called “pureSERMs”. The ideal SERM is a pure SERM of the type EM-652.HCl because ofits potent and pure antiestrogenic activity in the mammary gland.Others, like Raloxifene, Tamoxifen, Droloxifene, 4-hydroxy-Tamoxifen(1-(4-dimethylaminoethoxyphenyl)-1-(4-hydroxyphenyl)-2-phenyl-but-1-ene),Toremifene, 4-hydroxy-Toremifene[(Z)-(2)-2-[4-(4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine),LY 353 381, LY 335 563, GW-5638, Lasofoxifene, Idoxifene andBazedoxifene have some estrogenic activities in the breast andendometrium. This second series of SERMs may be called “mixed SERMs”.The unwanted estrogenic activities of these “mixed SERMs” may beinhibited by addition of pure “SERMs” as shown in FIGS. 5 and 6 in vitrotests and in FIG. 7 in an in vivo test of breast cancer. Since humanbreast carcinoma xenografts in nude mice are the closest available modelof human breast cancer, we have thus compared the effect of EM-800 andTamoxifen alone and in combination on the growth of ZR-75-1 breastcancer xenografts in nude mice.

The applicant believes that it is very important that SERMs of theinvention act as pure antiestrogens in breast, uterine, and endometrialtissues because SERMs have to counteract potential side-effects ofestrogens, particularly those formed from the exogenous precursors ofsex steroids which can increase the risk of cancer in these tissues.Particularly, the applicant believes that benzopyran derivatives of theinvention having the absolute configuration 2S at position 2 is moresuitable than its racemic mixture. Thus, in U.S. Pat. No. 6,060,503,optically active benzopyran antiestrogens having 2S configuration aredisclosed to treat estrogen-exacerbated breast and endometrial cancerand these compounds are shown to be significantly more efficient thanracemic mixtures (See FIGS. 1-5 of U.S. Pat. No. 060,503).

The enantiomer of 2S configuration being difficult to be industriallyobtained as a pure state, the applicant believes that less than 10%,preferably less than 5% and more preferably less than 2% by weight ofcontamination by the 2R enantiomer is preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of treatment with DHEA (10 mg, percutaneously,once daily) or EM-800 (75 μg, orally, once daily) alone or incombination for 9 months on serum triglyceride (A) and cholesterol (B)levels in the rat. Data are expressed as the means±SEM. **: P<0.01experimental versus respective control.

FIG. 2 shows the effect of 37-week treatment with increasing doses(0.01, 0.03, 0.1, 0.3, and 1 mg/kg) of EM-800 or Raloxifene administeredon total serum cholesterol levels in the ovariectomized rat. Comparisonis made with intact rats and ovariectomized animals bearing an implantof 17β-estradiol (E₂); **p<0.01, experimental versus OVX control rats.

FIG. 3 shows: A) Effect of increasing doses of DHEA (0.3 mg, 1.0 mg or3.0 mg) administered percutaneously twice daily on average ZR-75-1 tumorsize in ovariectomized (OVX) nude mice supplemented with estrone.Control OVX mice receiving the vehicle alone are used as additionalcontrols. The initial tumor size was taken as 100%. DHEA wasadministered percutaneously (p.c.) in a 0.02 ml solution of 50%ethanol-50% propylene glycol on the dorsal skin. B) Effect of treatmentwith increasing doses of DHEA or EM-800 (a SERM of the presentinvention) alone or in combination for 9.5 months on ZR-75-1 tumorweight in OVX nude mice supplemented with estrone. **, p<0.01, treatedversus control OVX mice supplemented with estrone.

FIG. 4 shows the effect of increasing oral doses of the antiestrogenEM-800 (15 μg, 50 μg or 100 μg) (B) or of percutaneous administration ofincreasing doses of DHEA (0.3, 1.0 or 3.0 mg) combined with EM-800 (15μg) or EM-800 alone (A) for 9.5 months on average ZR-75-1 tumor size inovariectomized (OVX) nude mice supplemented with estrone. The initialtumor size was taken as 100%. Control OVX mice receiving the vehiclealone were used as additional controls. Estrone was administeredsubcutaneously at the dose of 0.5 μg once daily while DHEA was dissolvedin 50% ethanol-50% propylene glycol and applied on the dorsal skin areatwice daily in a volume of 0.02 ml. Comparison is also made with OVXanimals receiving the vehicle alone.

FIG. 5 shows the effect of increasing concentrations of EM-800,(Z)-4-OH-Tamoxifen, (Z)-4-OH-Toremifene and Raloxifene on alkalinephosphatase activity in human Ishikawa cells. Alkaline phosphataseactivity was measured after a 5-day exposure to increasingconcentrations of indicated compounds in the presence or absence of 1.0nM E₂. The data are expressed as the means±SEM of four wells. When SEMoverlaps with the symbol used, only the symbol is shown (Simard, Sanchezet al., 1997).

FIG. 6 shows the blockade of the stimulatory effect of(Z)-4-OH-Tamoxifen, (Z)-4-OH-Toremifene, Droloxifene and Raloxifene onalkaline phosphatase activity by the antiestrogen EM-800 in humanIshikawa carcinoma cells. Alkaline phosphatase activity was measuredafter a 5-day exposure to 3 or 10 nM of the indicated compounds in thepresence or absence of 30 or 100 nM EM-800. The data are expressed asthe means±SD of eight wells with the exception of the control groupswere data are obtained from 16 wells (Simard, Sanchez et al., 1997).

FIG. 7 shows that the stimulatory effect of Tamoxifen on the growth ofhuman breast cancer ZR-75-1 xenografts is completely blocked bysimultaneous administration of EM-652.HCl. EM-652.HCl, by itself, inagreement with its pure antiestrogenic activity has no effect on tumorgrowth in the absence of Tamoxifen.

FIG. 8 shows the comparison of the effects of standard ERT (estrogen) orHRT (estrogen+progestin) and the combination of dehydroepiandrosteroneand the SERM Acolbifene on parameters of menopause. The addition ofAcolbifene to dehydroepiandrosterone will counteract the potentiallynegative effect of estrogen formed from dehydroepiandrosterone.

FIG. 9 shows sections of rat mammary gland:

-   -   a) Untreated animal. The lobules (L) consist of a few alveoli.        Insert. High magnification showing alveoli.    -   b) Animal treated with EM-800 (0.5 mg/kg, b w per day) for 12        weeks. The lobules (L) are reduced in size. Insert. High        magnification showing atrophied alveolar cells.

FIG. 10 shows sections of rat endometrium:

-   -   a) Untreated animal. The luminal epithelium (LE) is        characterized by columnar epithelial cells while the glandular        epithelium (GE) is rather cuboidal. The stroma contains several        cellular elements and collagen fibers.    -   b) Animal treated with EM-800 (0.5 mg/kg, b w per day) during 12        weeks. The luminal epithelium is markedly reduced in height. The        glandular epithelial cells have unstained cytophasm with no sign        of activity. The stroma is highly cellular due to reduction in        intercellular elements of the stroma.

FIG. 11 shows the effect on uterine weight of increasing concentrationsof EM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers)and Raloxifene administered orally for 9 days to ovariectomized micesimultaneously treated with estrone. *p<0.05, **p<0.01 versus E₁-treatedcontrol.

FIG. 12 shows the effect on vaginal weight of increasing concentrationsof EM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers)and Raloxifene administered orally for 9 days to ovariectomized micesimultaneously treated with estrone. **p<0.01 versus E₁-treated control.

FIG. 13 shows the effect on uterine weight of 1 μg and 10 μg ofEM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers)and Raloxifene administered orally for 9 days to ovariectomized mice.**p<0.01 versus OVX control.

FIG. 14 shows the effect on vaginal weight of 1 μg and 10 μg ofEM-652.HCl, Lasofoxifene (free base; active and inactive enantiomers)and Raloxifene administered orally for 9 days to ovariectomized mice.**p<0.01 versus OVX control.

FIG. 15 shows the effect of 12-month treatment withdehydroepiandrosterone (DHEA) alone or in combination with Flutamide orEM-800 on trabecular bone volume in ovariectomized rats. Intact animalsare added as additional controls. Data are presented as mean±SEM**p<0.01versus OVX Control.

FIG. 16 shows the effect of 12-month treatment withdehydroepiandrosterone (DHEA) alone or in combination with Flutamide orEM-800 on trabecular number in ovariectomized rats. Intact animals areadded as additional controls. Data are presented as mean±SEM**p<0.01versus OVX Control.

FIG. 17 shows proximal tibia metaphyses from intact control (A),ovariectomized control (B), and ovariectomized rats treated with DHEAalone (C) or in combination with Flutamide (D) or EM-800 (E). Note thereduced amount of trabecular bone (T) in ovariectomized control animals(B), and the significant increase in trabecular bone volume (T) inducedafter DHEA administration (C). The addition of Flutamide to DHEApartially blocked the effect of DHEA on the trabecular bone volume (D),whereas the combination of DHEA and EM-800 provided complete protectionagainst the ovariectomy-associated bone loss. Modified trichromeMasson-Goldner, magn.×80. T: Trabeculae, GP: Growth Plate.

FIG. 18 shows the effects of antiestrogens on ZR-75-1 tumor growth.Effect of treatment with 7 antiestrogens for 161 days, onestrone-induced growth of human ZR-75-1 breast tumors in ovariectomizednude mice. Tumor size is expressed as the percentage of initial tumorarea (Day 1=100%). Data is expressed as means±SEM (n=18-30tumors/group); ## p<0.01 vs EM-652.HCl; **p<0.01 vs OVX. Antiestrogenswere administered orally once daily at the dose of 50 μg/mouse underestrone stimulation obtained with subcutaneous 0.5-cm silastic implantscontaining 1:25 ratio of estrone and cholesterol.

FIG. 19 shows the effects of antiestrogens on ZR-75-1 tumor growth.Effect of treatment with 7 antiestrogens for 161 days, on the growth ofhuman ZR-75-1 breast tumors in ovariectomized nude mice. Tumor size isexpressed as the percentage of initial tumor area (Day 1=100%). Date isexpressed as means±SEM (n=18-30 tumors/group); ## p<0.01 vs EM-652.HCl;**p<0.01 vs OVX. Antiestrogens were administered orally once daily atthe dose of 100 μg/mouse in absence of estrogen stimulation.

FIG. 20 shows the effects of antiestrogens on ZR-75-1 tumor growth.Effect of treatment with the antiestrogens Tamoxifen, EM-652.HCl(Acolbifene) and the combination of Tamoxifen and EM-652.HCl for 161days, on the growth of human ZR-75-1 breast tumors in ovariectomizednude mice. Tumor size is expressed as the percentage of initial tumorarea (Day 1=100%). Data is expressed as means±SEM (n=18-30tumors/group); ##p<0.01 vs EM-652.HCl; **p<0.01 vs OVX. Antiestrogenswere administered orally once daily at the dose of 200n/mouse in absenceof estrogen stimulation.

FIG. 21 shows the effects of antiestrogens on categories of response.Effect of a 161-day administration of 7 antiestrogens, on the categoryof response of human ZR-75-1 breast tumors in ovariectomized nude mice.Complete regression identifies those tumors that were undetectable atthe end of treatment; partial regression corresponds to the tumors thatregressed ≥50% of their original size; stable response refers to tumorsthat regressed <50% or progressed ≤50%; and progression indicates thatthey progressed more than 50% compared with their original size.Antiestrogens were administered orally once daily at the dose of 50μg/mouse under estrone stimulation obtained with subcutaneous 0.5-cmsilastic implants containing 1:25 ratio of estrone and cholesterol.

FIG. 22 shows the effects of antiestrogen on categories of response.Effect of a 161-day administration of 7 antiestrogens, on the categoryof response of human ZR-75-1 breast tumors in ovariectomized nude mice.Complete regression identifies those tumors that were undetectable atthe end of treatment; partial regression corresponds to the tumors thatregressed ≥50% of their original size; stable response refers to tumorsthat regressed <50% or progressed ≤50%; and progression indicates thatthey progressed more than 50% compared with their original size.Antiestrogens were administered orally once daily at the dose of 200μg/mouse in absence of estrogen stimulation.

FIG. 23 shows the effects of antiestrogen on categories of response.Effect of a 161-day administration of the antiestrogens Tamoxifen,EM-652.HCl (Acolbifene) and the combination of Tamoxifen and EM-652.HCl,on the category of response of human ZR-75-1 breast tumors inovariectomized nude mice. Complete regression identifies those tumorsthat were undetectable at the end of treatment; partial regressioncorresponds to the tumors that regressed ≥50% of their original size;stable response refers to tumors that regressed <50% or progressed 50%;and progression indicates that they progressed more than 50% comparedwith their original size. Antiestrogens were administered orally oncedaily at the dose of 200 μg/mouse in absence of estrogen stimulation.

FIG. 24 is a Study Design Diagram of the phase II-III placebo-controlledstudy to evaluate the effects of DHEA on vasomotor symptoms (hotflushes) in postmenopausal women.

FIG. 25 shows the effect of a daily dose of DHEA or placebo on meannumber of moderate to severe hot flushes during 16 weeks of treatment(*, p<0.05 DHEA versus placebo).

FIG. 26 shows the treatment with a daily 50 mg dose of DHEA or placeboon mean number of all hot flushes (mild, moderate and severe) during 16weeks of treatment (*, p<0.05 DHEA versus placebo).

FIG. 27 shows the maturation index measured on Day 1 and Day 7 in 40-75year-old postmenopausal women following daily administration of vaginalsuppositories containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data areexpressed as means±SEM (n=9 or 10). *, p<0.05, **, p<0.01, Data on Day 7versus Data on Day 1.

FIG. 28 shows vaginal pH measured on Day 1 and Day 7 in 40-75 year oldpostmenopausal women following daily administration of vaginalsuppositories containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data areexpressed as means±SEM (n=9 or 10). *, p<0.05, **, p<0.01, Data on Day 7versus Data on Day 1.

DETAILED DESCRIPTION OF THE INVENTION Beneficial Effects of DHEA

The most widely recognized fact concerning menopause is that there is aprogressive decrease and finally an arrest of estrogen secretion by theovaries. The cessation of ovarian estrogen secretion is illustrated bythe marked decline in circulating 17β-estradiol (E₂) levels. This easilymeasurable change in circulating E₂ levels coupled with the beneficialeffects of estrogens on menopausal symptoms and bone resorption hasconcentrated most of the efforts of hormone replacement therapy onvarious forms of estrogens as well as to combinations of estrogen andprogestin in order to avoid the potentially harmful stimulatory effectsof estrogens used alone on the endometrium which can result inendometrial hyperplasia and cancer.

The rapid fall in circulating 17β-estradiol (E₂) at menopause, coupledwith the beneficial effects of exogenous estrogens on menopausalsymptoms and bone resorption has focused most of the efforts of hormonereplacement therapy on various forms of estrogens as well as tocombinations of estrogen and progestin in order to avoid the risk ofendometrial cancer induced by estrogens administered alone.

Hormone replacement therapy (HRT), estrogen and progestin, is used inpostmenopausal women for the acute symptoms arising from estrogendeficiency, particularly hot flushes and night sweats, and for the longterm prevention of osteoporosis and possibly cardiovascular disease.While progestins are effective at protecting the uterus from thestimulatory effects of long term estrogen exposure, it carries its ownside effects, in particular dysfunctional uterine bleeding (Archer etal., 1999). This is a frequent side effect and a common reason for womento prematurely stop hormone replacement therapy within the first 6-12months. The classical HRT has recently been seriously questioned or evenabandoned by many women following data indicating that the combinationof Premarin and Provera (Prempro) causes a 26% increase in the incidenceof breast cancer at 5.2 years of follow-up with a potential negativeimpact on cardiovascular events (Women's Health Initiative, 2002).

We feel that the increased understanding of androgen and estrogenformation and action in peripheral target tissues called intracrinology(Labrie, 1991; Labrie et al., 1992a; Labrie et al., 1992b; Labrie etal., 1994; Labrie et al., 1995; Luu-The et al., 1995a; Labrie et al.,1996; Labrie et al., 1997a; Labrie et al., 1997b; Labrie et al., 1997c;Labrie et al., 1997d) as well as our recent observations indicating thepredominant role of androgens over that of estrogens in the preventionof bone loss after ovariectomy in the rat (Martel et al., 1998) and theobservation of a similar situation in post-menopausal women (Labrie etal., 1997c) have paved the way for a timely and potentially highlysignificant progress in the field of sex steroid replacement therapy andaging. Such a possibility is well supported by our observations.

In Berger et al. (2005) it is shown particularly interesting effects ofDHEA on the three layers of the vaginal wall of rat vagina, namely ahighly mucified epithelium, an increased muscularis thickness andincreased collagen fiber compactness in the lamina propria. Thus DHEAexerts both androgenic and estrogenic effects on the vaginal mucosa,providing a more physiological replacement therapy.

The present invention is thus based upon the recent progress achieved inour understanding of sex steroid physiology in men and women (Labrie,1991; Labrie et al., 1992a; Labrie et al., 1992b; Labrie et al., 1994;Labrie et al., 1995a; Luu-The et al., 1995a; Labrie et al., 1997a;Labrie et al., 1997b; Labrie et al., 1997c; Labrie et al., 1997d) andthe recognition that women, at menopause, are not only deprived fromestrogens activity due to a declining ovarian activity, but have alreadybeen submitted for a few years to a decreasing exposure to androgens. Infact, normal women produce an amount of androgens equivalent to twothirds of the androgens secreted in men (Labrie et al., 1997a).

The pool of androgens in women decreases progressively from the age of30 years in parallel with the decrease in the serum concentration ofDHEA and DHEA-S (Labrie et al., 1997b). Consequently, it appears logicalto use both androgenic and estrogenic replacement therapy at peri- andpost-menopause, thus maintaining a physiological balance between thesetwo classes of sex steroids in each cell and tissue, a goal which canonly be met by the local formation of androgens and estrogens inperipheral tissues from the steroid precursor DHEA.

DHEA, a Predominant Source of Androgens Role of DHEA in Peripheral SexSteroid Formation

Humans, with some other primates, are unique among animal species inhaving adrenals that secrete large amounts of the inactive precursorsteroids DHEA and especially DHEA-S, which are converted into potentandrogens and/or estrogens in peripheral tissues. Plasma DHEA-S levelsin adult and women are 500 times higher than those of testosterone and10,000 times higher than those of estradiol, thus providing a largesupply of substrate for the formation of androgens and/or estrogens. Asmentioned above, the local synthesis and action of sex steroids inperipheral target tissues has been called intracrinology (Labrie et al.,1988; Labrie, 1991). Recent and rapid progress in this area has beenmade possible by the elucidation of the structure of most of thetissue-specific genes that encode the steroidogenic enzymes responsiblefor the transformation of DHEA-S and DHEA into androgens and/orestrogens locally in peripheral tissues (Labrie et al., 1992a; Labrie etal., 1992c; Labrie et al., 1995; Luu-The et al., 1995b; Labrie et al.,1996; Labrie et al., 1997d).

The major importance of DHEA and DHEA-S in human sex steroid physiologyis illustrated by the estimate that approximately 50% of total androgensin adult men derive from these adrenal precursor steroids (Labrie etal., 1985; Belanger et al., 1986; Labrie et al., 1993), while, in women,our best estimate of the intracrine formation of estrogens in peripheraltissues is in the order of 75% before menopause and close to 100% aftermenopause (Labrie, 1991).

The almost exclusive focus on the role of ovarian estrogens has removedthe attention from the dramatic 70% fall in circulating DHEA whichalready occurs between the ages of 20 to 30 and 40 to 50 years (Migeonet al., 1957; Vermeulen and Verdonck, 1976; Vermeulen et al., 1982;Orentreich et al., 1984; Belanger et al., 1994; Labrie et al., 1997b).Since DHEA is transformed to both androgens and estrogens in peripheraltissues, such a fall in serum DHEA and DHEA-S explains why women atmenopause, as mentioned above, are not only lacking estrogens but arealso deprived from androgens.

As mentioned above, recent data suggest that progestins have a negativeimpact on breast cancer (Clarke and Sutherland, 1990; Musgrove et al.,1991; Horwitz, 1992), with reports indicating an increased risk of thedisease (Colditz et al., 1995). In this context, it is important toindicate that the absence of a stimulatory effect of DHEA on the humanendometrium (Labrie et al., 1997c) eliminates the need to administer aprogestin to neutralize the potential effect of estrogens on theendometrium.

Concerning the breast, DHEA is known to prevent the development (Luo etal., 1997) and to inhibit the growth (Li et al., 1993) ofdimethylbenz(a)anthracene mammary tumors in the rat. DHEA, in addition,inhibits the growth of human breast cancer xenografts in nude mice (Seeexample 1 and Couillard et al., 1998). Thus, contrary to estrogens andprogestins which exert stimulatory effects, DHEA is expected to inhibitboth the development and the growth of breast cancer in women.

As well demonstrated in our previous studies, supplementation withphysiological amounts of exogenous DHEA permits the biosynthesis ofandrogens and estrogens only in the appropriate target tissues whichcontain the specific steroidogenic enzymes. The active androgens andestrogens thus synthesized remain in the cells of origin and very littleleakage occurs into the circulation. In fact, the most striking effectsof DHEA administration are on the circulating levels of the glucuronidederivatives of the metabolites of DHT, namely ADT-G and 3α-diol-G, thesemetabolites being produced locally in the peripheral intracrine tissueswhich possess the appropriate steroidogenic enzymes to synthesize DHTfrom the adrenal precursors DHEA and DHEA-S and, thereafter, to furthermetabolize DHT into inactive conjugates (Labrie, 1991; Labrie et al.,1996). This local biosynthesis and action of androgens in target tissueseliminates the exposure of other tissues to androgens and thus minimizesthe risks of undesirable masculinizing or other androgen-related sideeffects. The same applies to estrogens although we feel that a reliableparameter of total estrogen secretion (comparable to the glucuronidesfor androgens) is not yet available.

Role of Androgens and Estrogens in Bone Physiology

A predominant role of androgens on bone physiology is well documented(Labrie et al., 1997c; Martel et al., 1998). In fact, both testosteroneand DHT increased the transcription of a (I) procollagen mRNA inosteoblast-like osteosarcoma cells (Benz et al., 1991). Treatment withDHT has also been shown to stimulate endochondral bone development inthe orchiectomized rat (Kapur and Reddi, 1989). Moreover, bone mineraldensity measured in the lumbar spine, femoral trochanter and total bodywas increased more by estrogen+testosterone implants than by E₂ aloneover a 24-month treatment period in postmenopausal women (Davis et al.,1995).

Moreover, in established osteoporosis, anabolic steroids have beenreported to help prevent bone loss (Hennernan and Wallach, 1957).Similarly, subcutaneous E₂ and testosterone implants have been found tobe more efficient than oral estrogen in preventing osteoporosis inpostmenopausal women (Savvas et al., 1988). Although the differenceobserved in that study has been attributed to the different routes ofadministration of the estrogen, the cause of the difference could wellbe the action of testosterone. As index of increased bone formation, anincrease in serum osteocalcin, a marker of bone formation has been foundin postmenopausal women receiving methyltestosterone plus estrogen,compared with estrogen alone (Raisz et al., 1996). A similar stimulatoryeffect on serum osteocalcin has been observed following treatment ofpostmenopausal women with percutaneous DHEA for 12 months (Labrie etal., 1997c). Moreover, androgen therapy, as observed with nandrolonedecanoate, has been found to increase vertebral bone mineral density inpostmenopausal women (Need et al., 1989). Although androgens are gainingincreasing support due to their unique actions in postmenopausal women,virilizing effects are observed with the use of testosterone (Burger etal., 1984; Studd et al., 1987).

DHEA and Abdominal Obesity

Abdominal obesity is associated with an increased risk of insulinresistance, type 2 diabetes and atherosclerosis (Shimokata et al., 1989;Cefalu et al., 1995; Ferrannini et al., 1997; Kopelman, 2000). Amongother factors, hormonal changes, especially the declining secretion ofDHEA and DHEA-S by the adrenals is thought to be a factor involved(Tchernof et al., 1996). In rat and mouse models, DHEA administrationreduces visceral fat accumulation in diet-induced (Yen et al., 1977;Cleary and Zisk, 1986; Mohan et al., 1990; Hansen et al., 1997) obesity.A beneficial effect of DHEA has also been observed on the decrease ininsulin resistance that occurs with age (Han et al., 1998).

In a study performed in postmenopausal women who received a DHEA creamfor 12 months, we have found that insulin resistance was decreased whilesubcutaneous fat at the level of the thigh was also decreased (Diamondet al., 1996). Moreover, the daily administration of 50 mg DHEA for 6months in 65 to 78-year old men and women decreased abdominal visceralfat by 10.2% in women and 7.4% in men (Villareal and Holloszy, 2004). Inthe same study, abdominal subcutaneous fat was decreased by 6% in bothwomen and men. Moreover, the responsiveness of serum insulin to theglucose tolerance test was decreased by 13% with no change in theglucose response, thus leading to a 34% improvement in the insulinsensitivity index following DHEA administration. An improvement in DHEAaction has also been found in middle-aged men suffering fromhypercholesterolemia (Kawano et al., 2003).

In a previous study performed by the same group, DHEA administration for6 months decreased total body fat mass by 1.4 kg while fat-free mass wasincreased by 0.9 kg (Villareal et al., 2000). Effects of androgens onlibido, hot flushes and quality of life.

Community-based studies suggest self-reported sexual dysfunctions inwomen which range from 8% to 50%. In fact, low libido and sexualdysfunction increases with age in women from the third decade (Laumannet al., 1999) as well as after ovariectomy (Nathorst-Boos and vonSchoultz, 1992). While phychosocial and health factors are involved inlow arousal and sexual desire (Dennerstein et al., 1997) it is believedthat low androgens play an independent role (Bachmann et al., 2002;Miller et al., 2004).

Androgens are known to play a role in women's arousability, pleasure aswell as intensity and ease of orgasm. Androgens are also involved in theneurovascular smooth muscle response of swelling and increasedlubrication (Basson, 2004). Estrogens affect the vulval and vaginalcongestive responses. Since estrogens also affect mood, they have aninfluence on sexual interest (Basson, 2004). It should be rememberedthat DHEA is transformed into both androgens and estrogens in the vagina(Sourla et al., 1998) (Berger et al., 2005)

In addition, the detailed benefits of androgens added to ERT or HRT havebeen described on general well-being, energy, mood, and general qualityof life (Sherwin and Gelfand, 1985; Sherwin, 1988). Improvements in themajor psychologic and psychomatic symptoms, namely irritability,nervousness, memory, and insomnia have been observed following additionof androgens to estrogen replacement therapy (ERT) (Notelovitz et al.,1991).

Loss of libido and/or sexual satisfaction are common in earlypost-menopause. The addition of androgens to hormone replacement therapy(HRT) is known to have beneficial effects on these problems. Shifren etal., (2000) have found that transdermal testosterone administered bypatch improved sexual frequency, pleasure and mood in surgicallymenopausal women. The effect was seen at a daily 300 μg dose oftestosterone, a dose that led to serum testosterone levels in the upperlimit of normal. Testosterone treatment has also been studied in nonandrogen-deficient women complaining of decreased libido (Goldstat etal., 2003). Such treatment with testosterone improved libido, sexualfunction as well as quality of life compared to placebo. Similarly, inmenopausal women with normal levels of androgens, the addition ofmethyltestosterone to estrogen increased sexual desire and frequency ascompared to estrogen alone (Lobo et al., 2003). Among women withdysfunction of sexual interest, desire, androgen therapy has beensuggested for those having free serum testosterone levels within thelower quantile of the reference range (Bachmann et al., 2002). In fact,there is increased use of testosterone to treat hypoactive sexual desiredisorder (HSDD) (Sherwin and Gelfand, 1987; Davis et al., 1995; Shifrenet al., 2000; Goldstat et al., 2003). These randomized clinical trialsdemonstrate that testosterone is effective in women with HSDD.

The androgenic effect of DHEA should also be useful in reducing hotflushes. In fact, androgen therapy is successful in reducing hot flushesin hypogonadal men (De Fazio et al., 1984) and in menopausal transitionin women (Overlie et al., 2002). Moreover, the addition of androgens hasbeen found to be effective in relieving hot flushes in women who hadunsatisfactory results with estrogen alone (Sherwin and Gelfand, 1984).Hot flushes are one of the main reasons women initially seek HRTtherapy, and estrogen is very effective at alleviating this symptom.

A clear example of nature of androgen deficiency of adrenal origin isprovided by cases of adrenal insufficiency. (Arlt et al., 1999) havestudied the effect of DHEA, 50 mg daily and placebo for 4 months in apopulation of women suffering from adrenal insufficiency. Treatment withDHEA raised serum testosterone in the low normal range. Such treatmentincreased the frequency of sexual thoughts, interest and satisfaction.Well-being, depression and anxiety were also improved. In a study whereDHEA was administered at a high 300 mg daily dose, a greater subjectivemental (p<0.016) and physical (p<0.030) was observed in response to anerotic video (Hackbert and Heiman, 2002). In a study performed in womenreceiving 50 mg DHEA daily, improved libido was observed in women aged70 years or more but not in those aged 60 to 70 years (Baulieu, 1999).DHEA has also shown beneficial effects on hot flushes (Baulieu, 1999;Stomati et al., 2000). In a recent Canadian survey, 70.8% ofpractitioners add androgen to estrogen to enhance quality of life(Gelfand, 2004).

Other Potential Benefits of DHEA

The 70 to 95% reduction in the formation of DHEA and DHEA-S by theadrenals during aging results in a dramatic reduction in the formationof androgens and estrogens in peripheral target tissues, which couldwell be involved in the pathogenesis of age-related diseases such asinsulin resistance (Coleman et al., 1982; Schriock et al., 1988) andobesity (Nestler et al., 1988; MacEwen and Kurzman, 1991; Tchernof etal., 1995). Low circulating levels of DHEA-S and DHEA have, in fact,been found in patients with breast cancer (Zumoff et al., 1981) and DHEAhas been found to exert antioncogenic activity in a series of animalmodels (Schwartz et al., 1986; Gordon et al., 1987; Li et al., 1993).DHEA has also been shown to have immuno modulatory effects in vitro(Suzuki et al., 1991) and in vivo in fungal and viral diseases(Rasmussen et al., 1992), including HIV (Henderson et al., 1992). On theother hand, a stimulatory effect of DHEA on the immune system has beendescribed in postmenopausal women (Casson et al., 1993).

Previous Data Obtained with DHEA in Women

The use of estrogen replacement therapy requires the addition ofprogestins to counteract the endometrial proliferation induced byestrogens while both estrogens and progestins could increase the risk ofbreast cancer (Bardon et al., 1985; Colditz et al., 1995). In order toavoid the limitations of standard estrogen (ERT) or hormonal replacementtherapy (HRT), we have studied the effect of DHEA administration to 60to 70 year old women for 12 months on bone mineral density, parametersof bone formation and turnover, serum lipids, glucose and insulin,adipose tissue mass, muscular mass, energy, well-being as well as onvaginal and endometrial histology (Diamond et al., 1996; Labrie et al.,1997c). DHEA was administered percutaneously to avoid first passage ofthe steroid precursor through the liver.

We have thus evaluated the effect of chronic replacement therapy with a10% DHEA cream applied once daily for 12 months in 60 to 70 year oldwomen (N=15). Anthropometric measurements showed no change in bodyweight but a 9.8% decrease in subcutaneous skin fold thickness at 12months (p<0.05) (Diamond et al., 1996). Bone mass density was increasedby 2.3% at the hip, 3.75% at the hip Ward's triangle, and 2.2% at thelumbar spine level (all p<0.05). These changes in bone mineral densitywere accompanied by significant decreases at 12 months of 38% and 22% inurinary hydroxyproline and in plasma bone alkaline phosphatase,respectively (all p<0.05). An increase of 135% over control (p<0.05) inplasma osteocalcin was concomitantly observed, thus suggesting astimulatory effect of DHEA on bone formation.

Measurements of mid-thigh fat and muscle areas by computed tomographyhave shown a 3.8% decrease (p<0.05) of femoral fat and a 3.5% increase(p<0.05) in femoral muscular area at 12 months (Diamond et al., 1996).There was no significant change in abdominal fat measurements. Thesechanges in body fat and muscular surface areas were associated with a12% decrease (p<0.05) of fasting plasma glucose and a 17% decrease(p<0.05) in fasting plasma insulin levels. Treatment with DHEA had noundesirable effect on the lipid or lipoprotein profile. In fact, therewas an overall trend for a 3% to 10% decrease in total cholesterol andits lipoprotein fractions. Plasma triglycerides were not affected.

The index of sebum secretion was 79% increased after 12 months of DHEAtherapy with a return to pretreatment values 3 months after cessation oftreatment. DHEA administration stimulated vaginal epithelium maturationin 8 out of 10 women who had a maturation value of zero at the onset oftherapy while a stimulation was also seen in the three women who had anintermediate vaginal maturation before therapy. Most importantly, theestrogenic stimulatory effect observed in the vagina was not found inthe endometrium which remained completely atrophic in all women after 12months of DHEA treatment (Labrie et al., 1997c).

The present data clearly indicate the beneficial effects of DHEA therapyin postmenopausal women through its transformation into androgens and/orestrogens in specific intracrine target tissues without significant sideeffects. The absence of stimulation of the endometrium by DHEAeliminates the need for progestin replacement therapy, thus avoiding thefear of progestin-induced breast cancer. The observed stimulatory effectof DHEA on bone mineral density and the increase in serum osteocalcin, amarker of bone formation, are of particular interest for the preventionand treatment of osteoporosis and indicate a unique activity of DHEA onbone physiology, namely on bone formation while, ERT and HRT can onlyreduce the rate of bone loss.

A role of androgens has been proposed on depression, memory loss, lossof cognition and brain cell activity (Almeida et al., 2008, Azad et al.,2003 and Hajszan et al., 2008). Estrogens which can also be synthesizedin brain from DHEA have been shown to have a beneficial role inAlzheimer's disease, memory loss and loss of cognition (Rocca et al.,2007). Three metaanalyses have shown a 20 to 40% decreased risk ofAlzheimer's disease in women who used estrogen after menopause (Yaffe etal., 1998, Leblanc et al., 2001, Hogovorst et al., 2000). Estrogenreduces beta-amyloid deposition in the brain whereas progesterone hasthe opposite effect (Xu et al, 1998, Huang et al., 2004).

An association between lack of estrogen and cognitive impairment ordementia is supported by laboratory data. Among them estrogen improvessynapse formation on dendritic spines in the hippocampi ofoophorectomized rats (Mc Ewen and Alves, 1999, Monk and Brodatz, 2000).Moreover, estrogen improves cerebral blood flow and glucose metabolismand it may act as an antioxidant ((Mc Ewen and Alves, 1999; Monk andBrodatz, 2000; Gibbs and Aggamal, 1998). Estrogen has also been found toprevent B-Amyloid 1-42 from inducing a rise in intracellular calcium andfrom causing mitochondrial damage (Chen et al., 2006, Morrison et al.,2006).

There is now solid evidence from clinical studies that there is acritical age window for the beneficial effects of estrogens onneuroprotection (Rocca et al., 2007), cardiovascular disease (Manson etal., 2006) and overall mortality (Rocca et al., 2006). The best benefitsare seen when the treatment with E₂ has been started early withsometimes no or negative effects when the treatment is started lateafter menopause (WHI study). Estrogen reduces beta-amyloid deposition inthe brain whereas progesterone has the apposite effect (Xu et al., 1998,Huan et al., 2004).

Benefits of DHEA: Combination of Estrogen-Like and Androgenic Effects

It has been observed that androgens exert a direct antiproliferativeactivity on the growth of ZR-75-1 Androgens have also been shown toinhibit the growth of DMBA-induced mammary carcinoma in the rat, thisinhibition being reversed by the simultaneous administration of the pureantiandrogen Flutamide (Dauvois et al., 1989). Taken together, thesedata indicate the involvement of the androgen receptor in the inhibitoryaction of DHEA on breast cancer human breast cancer cells in vitro andthat such an inhibitory effect of androgens is additive to that of anantiestrogen (Poulin and Labrie, 1986; Poulin et al., 1988). Similarinhibitory effects have been observed in vivo on ZR-75-1 xenographts innude mice (Dauvois et al., 1991).

We have shown that DHEA exerts beneficial effects on bone in both thefemale rat (Luo et al., 1997), and postmenopausal women (Labrie et al.,1997c). Thus, in intact female rats, treatment with DHEA increases bonemineral density (BMD) of total skeleton, lumbar spine and femur (Luo etal., 1997).

The present invention is based upon the recent progress achieved in ourunderstanding of sex steroid physiology in women and the recognitionthat women, at menopause, are not only deprived from estrogen due to thearrest of estrogen secretion by the ovaries, but have already beensubmitted for a few years to a decreasing exposure to androgens. Infact, normal women produce an amount of androgens equivalent to twothirds of the androgens secreted in men (Labrie et al., 1997a). The poolof androgens in women decreases progressively from the age of 30 yearsin parallel with the decrease in the serum concentration of DHEA andDHEA-S (Labrie et al., 1997b). Consequently, it appears logical to useboth androgenic and estrogenic replacement therapy at peri- andpost-menopause, thus maintaining a physiological balance between thesetwo classes of sex steroids in each cell and tissue, a goal which canonly be met by the local formation of androgens and estrogens inperipheral tissues from a steroid precursor such as DHEA. The additionof a SERM like Acolbifene is to increase the positive effect on breastcancer protection as well as on other benefice of SERM administration.In FIG. 8, comparison is made with the positive and negative effects ofclassical ERT.

Previous data indicate the beneficial effects of DHEA therapy inpostmenopausal women through its transformation into androgens and/orestrogens in specific intracrine target tissues without significant sideeffects. In fact, our data obtained in the rat clearly demonstrate thatDHEA can provide the beneficial effects which are lacking with the useof a selective estrogen receptor modulator (SERM) alone.

Beneficial Effects of Acolbifene:

It can be seen in FIG. 7 that the approximately 100% stimulatory effectof Tamoxifen on tumor growth was completely blocked by simultaneoustreatment with EM-652 HCl. EM-652.HCl in accordance with its pureantiestrogenic activity did not exert any stimulatory effect on thegrowth of the human breast cancer ZR-75-1 xenografts in nude mice.

We have tested the steroidal antiestrogen fluvestrant (Faslodex, ICI182,780) and found it not to function as a SERM but antiestrogenfluvestrant may also be used in combination with DHEA in the presentinvention for the prevention of breast cancer. SERMs, in accordance withthe invention, may be administered in the same dosage as known in theart, even where the art uses them as antiestrogens instead of as SERMs.

We have also noted a correlation between the beneficial effect of SERMshave on serum cholesterol and beneficial estrogenic or estrogen-likeeffects on bone. SERMs have also a beneficial effect on hypertension,insulin resistance, diabetes, and obesity (especially abdominalobesity). Without intending to be bound by theory, it is believed thatSERMs, many of which preferably have two aromatic rings linked by one totwo carbon atoms, are expected to interact with the estrogen receptor byvirtue of the foregoing portion of the molecule that is best recognizedby the receptor. Preferred SERMs have side chains which may selectivelycause antagonistic properties in breast and usually uterine tissueswithout having significant antagonistic properties in other tissues.Thus, the SERMs may desirably functions as antiestrogens in the breastwhile surprisingly and desirably functioning as estrogens (or providingestrogen-like activity) in bone and in the blood (where concentrationsof lipid and cholesterol are favorably affected). The favorable effecton cholesterol and lipids translates to a favorable effect againstatherosclerosis which is known to be adversely, affected by improperlevels of cholesterol and lipids.

As demonstrated in FIG. 9, although circulating levels of 17β-estradiolwere elevated from 95.9±32.4 pg/ml in intact animals to 143.5±7.8 pg/ml(50% elevation in animals treated with EM-800, 0.5 mg/kg, orallydaily/for 12 weeks), a marked atrophy of the mammary gland was observed.Similarly, in FIG. 10, a marked atrophy of the endometrium was observedin animals receiving EM-800 (0.5 mg/kg). In these intact animalsreceiving the pure antiestrogen EM-800, the inhibitory effect ofestrogens at the hypothalamo-pituitary level was removed, thus causingincreased LH and then secondarily increased 17β-estradiol secretion bythe ovaries.

Hot flushes, cardiovascular symptoms, Alzheimer's disease, loss ofcognitive functions and insomnia involve certainly estrogen receptorssituated in the nervous central system. Probably, low levels ofestrogens in the brain, can explain at least in part, these conditions.Exogenous estrogens and particularly those (i.e. estradiol) formed bythe administration of sex steroid precursors can pass through the brainbarrier and bind to the estrogen receptor to restore the normalestrogenic action. On the other hand, SERMs of the invention, and moreparticularly those of Acolbifene family, cannot pass through the brainbarrier as shown in example 8. Thus, they cannot antagonise the positiveeffect of estrogens in brain but they antagonise the negative effects ofestrogens in the breast, uterine, and endometrial tissues rending thiscombination (SERM+sex steroid precursor) particularly attractive for thetreatment or reduction of the risk of acquiring the above-mentionedconditions.

As mentioned earlier, a role for androgens has also been suggested forall these symptoms. In fact, DHEA can provide both estrogens andandrogens in the brain according to physiological needs.

Overall Additive Benefits of Combining a Sex Steroid Precursor and aSERM or an Antiestrogen

The main reason why women consult their physician at menopause is theoccurrence of hot flushes, a problem well known to be eliminated byestrogen replacement therapy. Since the site responsible for hot flushesis the central nervous system (CNS) and EM-652 has very pooraccessibility to the CNS (data enclosed), it is expected that sexsteroid precursor administration will increase estrogen concentration incentral nervous system and thus will control hot flushes withoutinterference by the SERM. On the other hand, the SERM will eliminate allthe negative effects of estrogens at other sites, specially the risk ofbreast and uterine cancer. In fact, the addition of EM-652 to sexsteroid precursor blocks the stimulatory effect of formed estrogens onthe mammary gland and uterus while, in other tissues, EM-652 will exertits own beneficial effect, for example on the bone, where it partiallyreverses the effect of ovariectomy on bone mineral density.

By removing E2, we decrease the risk of breast cancer since our datashow that DHEA can decrease hot flushes, vasomotor symptoms and nightsweats. However, DHEA can be slightly transformed into estrogens, thusthe need for a SERM.

No adverse effect of EM-652 is seen on any parameter while it shouldexert marked beneficial effects for the prevention and treatment ofbreast and uterine cancer.

Preferred SERMs or antiestrogens discussed herein relate: (1) to alldiseases stated to be susceptible to the invention; (2) to boththerapeutic and prophylactic applications; and (3) to preferredpharmaceutical compositions and kits.

A patient in need of treatment or of reducing the risk of onset of agiven disease is one who has either been diagnosed with such disease orone who is susceptible of acquiring such disease.

Except where otherwise stated, the preferred dosage of the activecompounds (concentrations and modes of administration) of the inventionis identical for both therapeutic and prophylactic purposes. The dosagefor each active component discussed herein is the same regardless of thedisease being treated (or of the disease whose likelihood of onset isbeing reduced).

Except when otherwise noted or where apparent from context, dosagesherein refer to weight of active compounds unaffected by pharmaceuticalexcipients, diluents, carriers or other ingredients, although suchadditional ingredients are desirably included, as shown in the examplesherein. Any dosage form (capsule, pill, tablet, injection or the like)commonly used in the pharmaceutical industry is appropriate for useherein, and the terms “excipient”, “diluent”, or “carrier” include suchnonactive ingredients as are typically included, together with activeingredients in such dosage forms in the industry. For example, typicalcapsules, pills, enteric coatings, solid or liquid diluents orexcipients, flavorants, preservatives, or the like may be included.

All of the active ingredients used in any of the therapies discussedherein may be formulated in pharmaceutical compositions which alsoinclude one or more of the other active ingredients. Alternatively, theymay each be administered separately but sufficiently simultaneous intime so that a patient eventually has elevated blood levels or otherwiseenjoys the benefits of each of the active ingredients (or strategies)simultaneously. In some preferred embodiments of the invention, forexample, one or more active ingredients are to be formulated in a singlepharmaceutical composition. In other embodiments of the invention, a kitis provided which includes at least two separate containers wherein thecontents of at least one container differs, in whole or in part, fromthe contents of at least one other container with respect to activeingredients contained therein.

Combination therapies discussed herein also include use of one activeingredient (of the combination) in the manufacture of a medicament forthe treatment (or risk reduction) of the disease in question where thetreatment or prevention further includes another active ingredient ofthe combination in accordance with the invention. For example in oneembodiment, the invention provides the use of a SERM in the preparationof a medicament for use, in combination with a sex steroid precursor invivo, in the treatment of any of the diseases for which the presentcombination therapy is believed effective (i.e. hot flushes, sweat,irregular menstruation, and any symptoms related to menopause).

Estrogens are well-known to stimulate the proliferation of breastepithelial cells and cell proliferation itself is thought to increasethe risk of cancer by accumulating random genetic errors that may resultin neoplasia (Preston Martin et al., 1990). Based on this concept,antiestrogens have been introduced to prevent breast cancer with theobjective of reducing the rate of cell division stimulated by estrogens.

We have also studied the potential interaction of the inhibitory effectof the novel antiestrogen (EM-800) with that of sex steroid precursor(DHEA) on the growth of human ZR-75-1 breast cancer xenografts in nudemice by combined administration of the two drugs. FIGS. 3 and 4 showthat DHEA, by itself, at the doses used, causes a 50 to 80% inhibitionof tumor growth while the near complete inhibition of tumor growthachieved with a low dose of the antiestrogen was not affected by DHEA.

The limitations of bone mineral density (BMD) measurements are wellknown. As an example, BMD measurements showed no change in rats treatedwith the steroidal antiestrogen ICI 182780 (Wakeling, 1993) whileinhibitory changes were seen by histomorphometry (Gallagher et al.,1993). Similar differences were reported with Tamoxifen (Jordan et al.,1987; Sibonga et al., 1996).

It should be indicated that reduced bone mineral density is not the onlyabnormality associated with reduced bone strength. It is thus importantto analyze the changes in biochemical parameters of bone metabolisminduced by various compounds and treatments in order to gain a betterknowledge of their action.

It is particularly important to indicate that the combination of DHEAand EM-800 exerted unexpected beneficial effects on importantbiochemical parameters of bone metabolism. In fact, DHEA alone did notaffect the urinary hydroxyproline/creatinine ratio, a marker of boneresorption. Moreover, no effect of DHEA could be detected on dailyurinary calcium or phosphorus excretion (Luo et al., 1997). EM-800decreased the urinary hydroxyproline/creatinine ratio by 48% while,similarly to DHEA, no effect of EM-800 was seen on urinary calcium orphosphorus excretion. EM-800, moreover, had no effect on serum alkalinephosphatase activity, a marker of bone formation while DHEA increasedthe value of the parameter by about 75% (Luo et al., 1997).

One of the unexpected effects of the combination of DHEA and EM-800relates to the urinary hydroxyproline/creatinine ratio, a marker of boneresorption, which was reduced by 69% when both DHEA and EM-800 werecombined, this value being statistically different (p<0.01) from the 48%inhibition achieved by EM-800 alone while DHEA alone did not show anyeffect. Thus, the addition of DHEA to EM-800 increases by 50% theinhibitory effect of EM-800 on bone reabsorption. Most importantly,another unexpected effect of the addition of DHEA to EM-800 was theapproximately 84% decrease in urinary calcium (from 23.17±1.55 to3.71±0.75 μmol/24 h/100 g (p<0.01) and the 55% decrease in urinaryphosphorus (from 132.72±6.08 to 59.06±4.76 μmol/24 h/100 g (p<0.01)respectively, (Luo et al., 1997).

Importantly, the combination of EM-800 and DHEA in ovariectomized ratstreated for 12 months had beneficial effects on bone morphometry.Trabecular bone volume is particularly important for bone strength andto prevent bone fractures. Thus, in the above-mentioned study,trabecular bone volume of the tibia increased from 4.1±0.7% inovariectomized rats to 11.9±0.6% (p<0.01) with DHEA alone while theaddition of EM-800 to DHEA further increased trabecular bone volume to14.7±1.4%, a value similar to that found in intact controls (FIG. 15).

From a value of 0.57±0.08 per mm in ovariectomized rats, treatment withDHEA resulted in a 137% increase in trabecular bone number compared toovariectomized controls. The stimulatory effect of DHEA thus reached1.27±0.1 per mm while simultaneous treatment with EM-800 and DHEAresulted in an additional 28% increase in trabecular bone number(p<0.01) compared to that achieved by DHEA alone (FIG. 16). Similarly,the addition of EM-800 to DHEA treatment, resulted in an additional 15%(p<0.05) decrease in trabecular bone separation, compared to thatachieved with DHEA alone, thus leading to values not different fromthose seen in intact controls.

As complement to the numerical data presented in FIGS. 15,16, FIG. 17illustrates the increase in trabecular bone volume in the proximal tibiametaphysis induced by DHEA in ovariectomized treated animals (C)compared to ovariectomized controls (B), as well as the partialinhibition of the stimulatory effect of DHEA after the addition ofFlutamide to DHEA treatment (D). On the other hand, administration ofDHEA in combination with EM-800 resulted in a complete prevention of theovariectomy-induced osteopenia (E), the trabecular bone volume beingcomparable to that seen in intact controls (A).

TABLE 1 URINE SERUM CALCIUM PHOSPHORUS HP/Cr tALP GROUP (μmol/24 h/100g) (μmol/24 h/100 g) (μmol/mmol) (IU/L) CONTROL 23.17 ± 1.55 132.72 ±6.08 13.04 ± 2.19 114.25 ± 14.04 DHEA (10 mg) 25.87 ± 3.54 151.41 ±14.57 14.02 ± 1.59 198.38 ± 30.76* EM-800 (75 μg) 17.44 ± 4.5 102.03 ±25.13  6.81 ± 0.84** 114.11 ± 11.26 DHEA + EM-800  3.71 ± 0.75**  59.06± 4.76**  4.06 ± 0.28** 204.38 ± 14.20**

TABLE 2 Effect of 12-month treatment with dehydroepiandrosterone (DHEA)administered alone or in combination with Flutamide (FLU) or EM-800 onbone markers and serum lipids. Alkaline OH-proline/ phosphatasecreatinin Cholesterol Triglycerides Group IU/L μmol/mmol mmol/L mmol/LIntact Control  30 ± 3** 15.4 ± 1.3 2.28 ± 0.12 1.4 ± 0.2 OVX Control 51 ± 4 11.7 ± 1.2 2.29 ± 0.16 1.1 ± 0.1 OVX + DHEA 201 ± 25**  7.3 ±1.0* 1.78 ± 0.16* 0.8 ± 0.1 OVX + DHEA + FLU 103 ± 10** 14.5 ± 1.2 2.27± 0.15 0.8 ± 1.0 OVX + DHEA + EM-800 202 ± 17**  6.4 ± 1.0** 0.63 ±0.09** 1.0 ± 0.2 *p < 0.05; **p < 0.01 versus OVX Control

The importance of the androgenic component of the stimulatory effect ofDHEA on bone histomorphometry is also supported by the effect of DHEA onmarkers of bone formation and resorption. The concentration of serumalkaline phosphatase, a marker of bone formation (Meunier et al. 1987,Lauffenburger et al. 1977), was increased from 51±4 IU/L in OVX controlsto 201±25 IU/L in DHEA-treated animals, suggesting a stimulatory effectof DHEA on bone formation (Table 2). FLU reversed by 65% the stimulatoryeffect of DHEA on this parameter while EM-800 had no significant effect.On the other hand, since hydroxyproline released during collagendegradation is not reutilized in collagen synthesis, it is a usefulmarker of collagen metabolism or osteoclastic bone resorption. In thepresent study, the urinary hydroxyproline/creatinine ratio decreasedfrom 11.7±1.2 μmol/mmol in OVX controls to 7.3±1.0 μmol/mmol (p<0.05) inDHEA-treated rats (Table 2). The administration of FLU completelyprevented the inhibitory effect of DHEA on this parameter while EM-800had no statistically significant influence on the effect of DHEA.

Moreover, serum cholesterol was reduced by 22% from 2.29±0.16 to1.78±0.16 mmol/1(p<0.05) by DHEA treatment, an effect neutralized byconcomitant treatment with the pure antiandrogen FLU. The addition ofthe pure antiestrogen EM-800, on the other hand, decreased total serumcholesterol further to 0.63±0.09 mmol/1(p<0.01), thus reaching a 65%inhibitory effect. No statistically significant change was observed inserum triglyceride levels with any of the treatments used (Table 2).

It is also of interest to note that the potent inhibitory effect ofEM-800 on serum cholesterol is not prevented by simultaneous treatmentwith DHEA (Luo et al., 1997).

The bone loss observed at menopause in women is believed to be relatedto an increase in the rate of bone resorption which is not fullycompensated by the secondary increase in bone formation. In fact, theparameters of both bone formation and bone resorption are increased inosteoporosis and both bone resorption and formation are inhibited byestrogen replacement therapy. The inhibitory effect of estrogenreplacement on bone formation is thus believed to result from a coupledmechanism between bone resorption and bone formation, such that theprimary estrogen-induced reduction in bone resorption entrains areduction in bone formation (Parfitt, 1984).

Cancellous bone strength and subsequent resistance to fracture do notonly depend upon the total amount of cancellous bone but also on thetrabecular microstructure, as determined by the number, size, anddistribution of the trabeculae. The loss of ovarian function inpostmenopausal women is accompanied by a significant decrease in totaltrabecular bone volume (Melsen et al., 1978; Vakamatsou et al., 1985),mainly related to a decrease in the number and, to a lesser degree, inthe width of trabeculae (Weinstein and Hutson, 1987).

In order to facilitate the combination therapy aspect of the invention,for any indication discussed herein, the invention contemplatespharmaceutical compositions which include the SERM and the sex steroidprecursor in a single composition for simultaneous administration. Thecomposition may be suitable for administration in any traditional mannerincluding but not limited to oral administration, subcutaneousinjection, intramuscular injection or percutaneous administration. Inother embodiments, a kit is provided wherein the kit includes one ormore SERM and sex steroid precursor in separate or in one container. Thekit may include appropriate materials for oral administration, e.g.tablets, capsules, syrups and the like and for transdermaladministration, e.g., ointments, lotions, gels, creams, sustainedrelease patches and the like.

Applicants believe that administration of SERMs or antiestrogens and sexsteroid precursors has utility in the treatment and/or reduction of theincidence of hot flushes and sweat. The active ingredients of theinvention (whether SERM, antiestrogen or precursor or otherwise) may beformulated and administered in a variety of ways. When administeredtogether in accordance with the invention, the active ingredients may beadministered simultaneously or separately.

Active ingredient for transdermal or transmucosal is preferably from0.01% to 1%, DHEA or 5-diol. Alternatively, the active ingredient may beplaced into a vaginal ring or a transdermal patch having structuresknown in the art, for example, structures such as those set forth inE.P. Patent No. 0279982 or in an intravaginal cream, gel, ovule, orsuppository.

When formulated as an ointment, lotion, gel, cream, ovule, orsuppository or the like, the active compound is admixed with a suitablecarrier which is compatible with human skin or mucosa and which enhancestransdermal or transmucosal penetration of the compound through the skinor mucosa. Suitable carriers are known in the art and include but arenot limited to Klucel HF and Glaxal base. Some are commerciallyavailable, e.g., Glaxal base available from Glaxal Canada LimitedCompany. Other suitable vehicles can be found in Koller and Buri, S.T.P.Pharma 3(2), 115-124, 1987. The carrier is preferably one in which theactive ingredient(s) is (are) soluble at ambient temperature at theconcentration of active ingredient that is used. The carrier should havesufficient viscosity to maintain the inhibitor on a localized area ofskin or mucosa to which the composition has been applied, withoutrunning or evaporating for a time period sufficient to permitsubstantial penetration of the precursor through the localized area ofskin or mucosa and into the bloodstream where it will cause a desirableclinical effect. The carrier is typically a mixture of severalcomponents, e.g. pharmaceutically acceptable solvents and a thickeningagent. A mixture of organic and inorganic solvents can aid hydrophylicand lipophylic solubility, e.g. water and an alcohol such as ethanol.

When formulated as an ovule or a vaginal suppository or the like, theactive compound is admixed with a suitable carrier which is compatiblewith human vaginal mucosa. Preferred carriers are hard fats (mixture ofglycerides of saturated fatty acids), particularly Witepsol, andspecially Witepsol H-15 base (available from Medisca, Montreal, Canada).Any other lipophilic base such as Fattibase, Wecobee, cocoa butter,theobroma oil or other combinations of Witepsol bases could used.

Preferred sex steroid precursors are dehydroepiandrosterone (DHEA)(available, for example, from Proquina, Orizaba, Veracruz, Mexico).

The carrier may also include various additives commonly used inointments, lotions and suppositories and well known in the cosmetic andmedical arts. For example, fragrances, antioxidants, perfumes, gellingagents, thickening agents such as carboxymethylcellulose, surfactants,stabilizers, emollients, coloring agents and other similar agents may bepresent.

Treatment in accordance with the invention is suitable for indefinitecontinuation. The SERM or antiestrogenic compound and the sex steroidprecursor can also be administered, by the oral route, and may beformulated with conventional pharmaceutical excipients, e.g. spray driedlactose, microcrystalline cellulose, and magnesium stearate into tabletsor capsules for oral administration.

The active substances can be worked into tablets or dragee cores bybeing mixed with solid, pulverulent carrier substances, such as sodiumcitrate, calcium carbonate or dicalcium phosphate, and binders such aspolyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly byadding also lubricants such as magnesium stearate, sodium laurylsulfate, “Carbowax” or polyethylene glycol. Of course, taste-improvingsubstances can be added in the case of oral administration forms.

As further forms, one can use plug capsules, e.g. of hard gelatin, aswell as closed soft-gelatin capsules comprising a softener orplasticizer, e.g. glycerin. The plug capsules contain the activesubstance preferably in the form of granulate, e.g. in mixture withfillers, such as lactose, saccharose, mannitol, starches, such as potatostarch or amylopectin, cellulose derivatives or highly dispersed silicicacids. In solf-gelatin capsules, the active substance is preferablydissolved or suspended in suitable liquids, such as vegetable oils orliquid polyethylene glycols.

The lotion, ointment, gel or cream should be thoroughly rubbed into theskin so that no excess is plainly visible, and the skin should not bewashed in that region until most of the transdermal penetration hasoccurred preferably at least 4 hours and, more preferably, at least 6hours.

A transdermal patch may be used to deliver precursor in accordance withknown techniques. It is typically applied for a much longer period,e.g., 1 to 4 days, but typically contacts active ingredient to a smallersurface area, allowing a slow and constant delivery of activeingredient.

A number of transdermal drug delivery systems that have been developed,and are in use, are suitable for delivering the active ingredient of thepresent invention. The rate of release is typically controlled by amatrix diffusion, or by passage of the active ingredient through acontrolling membrane.

Mechanical aspects of transdermal devices are well known in the rat, andare explained, for example, in U.S. Pat. Nos. 5,162,037, 5,154,922,5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444, 4,568,343,5,064,654, 5,071,644, 5,071,657, the disclosures of which areincorporated herein by reference. Additional background is provided byEuropean Patent 0279982 and British Patent Application 2185187.

The device may be any of the general types known in the art includingadhesive matrix and reservoir-type transdermal delivery devices. Thedevice may include drug-containing matrixes incorporating fibers whichabsorb the active ingredient and/or carrier. In a reservoir-type device,the reservoir may be defined by a polymer membrane impermeable to thecarrier and to the active ingredient.

In a transdermal device, the device itself maintains active ingredientin contact with the desired localized skin surface. In such a device,the viscosity of the carrier for active ingredient is of less concernthan with a cream or gel. A solvent system for a transdermal device mayinclude, for example, oleic acid, linear alcohol lactate and dipropyleneglycol, or other solvent systems known in the art. The active ingredientmay be dissolved or suspended in the carrier.

For attachment to the skin, a transdermal patch may be mounted on asurgical adhesive tape having a hole punched in the middle. The adhesiveis preferably covered by a release liner to protect it prior to use.Typical material suitable for release includes polyethylene andpolyethylene-coated paper, and preferably silicone-coated for ease ofremoval. For applying the device, the release liner is simply peeledaway and the adhesive attached to the patient's skin. In U.S. Pat. No.5,135,480, the disclosure of which is incorporated by reference, Bannonet al., describe an alternative device having a non-adhesive means forsecuring the device to the skin.

It is necessary only that SERM, antiestrogen and sex steroid precursorbe administered in a manner and at a dosage sufficient to allow bloodserum concentration of each to obtain desired levels. In accordance withthe combination therapy of the invention, concentration of the SERM ismaintained within desired parameters at the same time that sex steroidprecursor concentration is maintained within desired parameters

One preferred sex steroid precursor is DHEA, although DHEA-S and analogsdiscussed below are also especially effective for the reasons statedbelow.

A selective estrogen receptor modulator of the invention has a molecularformula with the following features: a) two aromatic rings spaced by 1to 2 intervening carbon atoms, both aromatic rings being eitherunsubstituted or substituted by a hydroxyl group or a group converted invivo to hydroxyl; and b) a side chain possessing an aromatic ring and atertiary amine function or salt thereof.

One preferred SERM of the invention is Acolbifene:

Acolbifene (also called EM-652.HCl; EM-1538) is the hydrochloride saltof the potent antiestrogen EM-652. It is disclosed in U.S. Pat. No.6,710,059 B1. Another preferred SERM is Lasoxifene (Oporia; CP-336,156;(−)-cis-(5R,6S)-6-phenyl-5-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol,D-(−)-tartrate salt) (available from Pfizer Inc., USA).

Another preferred SERM is Bazedoxifene (TSE 424; WAY-TSE 424; WAY140424;1-[[4-[2-(hexahydro-1H-azepin-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol,acetate) developed by Wyeth Ayers (USA) and disclosed in JP10036347(American home products corporation) and approved in USA for theprevention of postmenopausal osteoporosis and non-steroidal estrogenderivatives described in WO 97/32837. Other preferred SERMs of theinvention include Tamoxifen ((Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine) (available from Zeneca, UK), Toremifene((Z)-2-[4-(4-Chloro-1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine)available from Orion, Finland, under the trademark Fareston orSchering-Plough), Droloxifene ((E)-3-[1-[4-[2-(Dimethylamino) ethoxy]phenyl]-2-phenyl-1-butenyl] phenol) and, from Eli Lilly and Co., USA:Raloxifene ([2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1-piperidinyl) ethoxy] phenyl]-methanone hydrochloride), LY335124, LY 326315, LY 335563 (6-hydroxy-3-[4-[2-(1-piperidinyl) ethoxy]phenoxyl]-2-(4-hydroxyphenyl) benzo[b]thiopene hydrochloride) andArzoxifene (LY 353381,6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxyl]-2-(4-methoxyphenyl)benzo [b] thiophene hydrochloride). Other preferred SERMs are Idoxifene((E)-1-[2-[4-[1-(4-Iodophenyl)-2-phenyl-1-butenyl]phenoxy]ethyl]pyrrolidine)(SmithKline Beecham, USA), Levormeloxifene(3,4-trans-2,2-dimethyl-3-phenyl-4-[4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxychroman)(Novo Nordisk, A/S, Denmark) which is disclosed in Shalmi et al. WO97/25034, WO 97/25035, WO 97/25037, WO 97/25038; and Korsgaard et al. WO97/25036), GW5638 (described by Willson et al., 1997) and indolederivatives (disclosed by Miller et al., EP 0802183A1) Are alsoincluded, Iproxifen (TAT 59;(E)-4-[1-[4-[2-(dimethylamino)ethoxy]phenyl]-2-[4-(1-methylethyl)phenyl]-1-butenyl]phenoldihydrogen phosphate) from Taiho (Japan), Ospemifene (FC 1271;((Z)-2-[4-(4-chloro-1,2-diphenyl-1-butenyl)phenoxyl]ethanol) fromavailable from Orion-Farmos Pharmaceutica, Finland, SERM 3471, HMR 3339and HMR 3656 from Sanofi-Aventis (France), SH 646 from Schering AG,Germany, Pipendoxifene (ERA 923) developed by Wyeth-Ayers, nonsteroidalestrogen derivatives described in WO 97/3283, Fispemifene developed byQuatRx (USA) and CC 8490 developed by Celgene in USA.

Any SERM used as required for efficacy, as recommended by themanufacturer, can be used. Appropriate dosages are known in the art. Anyother non steroidal antiestrogen commercially available can be usedaccording to the invention. Any compound having activity similar toSERMs (example: Raloxifene can be used).

SERMs administered in accordance with the invention are preferablyadministered in a dosage range between 0.01 to 10 mg/kg of body weightper day (preferably 0.05 to 1.0 mg/kg), with 5 mg per day, especially 10mg per day, in two equally divided doses being preferred for a person ofaverage body weight when orally administered, or in a dosage rangebetween 0.003 to 3.0 mg/kg of body weight per day (preferably 0.015 to0.3 mg/ml), with 1.5 mg per day, especially 3.0 mg per day, in twoequally divided doses being preferred for a person of average bodyweight when parentally administered (i.e. intramuscular, subcutaneous orpercutaneous administration). Preferably the SERMs are administeredtogether with a pharmaceutically acceptable diluent or carrier asdescribed below.

One preferred antiestrogen of the invention is fulvestrant (Faslodex;ICI 1827807α-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)-triene-3,17β-diol)which is intramuscularly administered with the dosage of 250 mg permonth available from AstraZeneca Canada Inc., Mississauga, Ontario,Canada.

With respect to all of the dosages recommended herein, the attendingclinician should monitor individual patient response and adjust dosageaccordingly.

EXAMPLES Example 1

In the mammary gland, androgens are formed from the precursor steroiddehydroepiandrosterone (DHEA). Clinical evidence indicates thatandrogens have inhibitory effects on breast cancer. Estrogens, on theother hand, stimulate the development and growth of breast cancer. Westudied the effect of DHEA alone or in combination with the newlydescribed pure antiestrogen, EM-800, on the growth of tumor xenograftsformed by the human breast cancer cell line ZR-75-1 in ovariectomizednude mice.

Mice received daily subcutaneous injections of 0.5 μg estrone (anestrogenic hormone) immediately after ovariectomy. EM-800 (15, 50 or 100μg) was given orally once daily. DHEA was applied twice daily (totaldose 0.3, 1.0 or 3.0 mg) to the dorsal skin either alone or incombination with a 15 μg daily oral dose of EM-800. Changes in tumorsize in response to the treatments were assessed periodically inrelation to the measurements made on the first day. At the end of theexperiments, tumors were dissected and weighed.

A 9.4-fold increase in tumor size in 9.5 months was observed inovariectomized mice receiving estrone alone in comparison with mice notreceiving estrone. Administration of 15, 50 or 100 μg EM-800 inestrone-supplemented ovariectomized led to inhibitions of 88%, 93%, and94% in tumor size, respectively. DHEA, on the other hand, at doses of0.3, 1.0 or 3.0 mg inhibited terminal tumor weight by 67%, 82%, and 85%,respectively. Comparable inhibitions in tumor size were obtained with adaily 15 μg oral dose of EM-800 with or without different doses ofpercutaneous DHEA. DHEA and EM-800 independently suppressed the growthof estrone-stimulated ZR-75-1 mouse xenograft tumors in nude mice.Administration of DHEA at the defined doses does not alter theinhibitory effect of EM-800.

Materials and Methods ZR-75-1 Cells

ZR-75-1 human breast cancer cells were obtained from the American TypeCulture Collection (Rockville, Md.) and routinely cultured as monolayersin RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate, 100 IU penicillin/ml, 100 μg streptomycin/ml, and 10% fetalbovine serum, under a humidified atmosphere of 95% air/5% CO₂ at 37° C.as described (Poulin and Labrie, 1986; Poulin et al., 1988). Cells werepassaged weekly after treatment with 0.05% trypsin: 0.02% EDTA (w/v).The cell cultures used for the experiments described in this report werederived from passage 93 of the cell line ZR-75-1.

Animals

Female homozygous Harlan Sprague-Dawley (nu/nu) athymic mice (28- to42-day-old) were obtained from HSD (Indianapolis, Ind., USA). Mice werehoused in vinyl cages with air filter tops in laminar air flow hoods andmaintained under pathogen-limited conditions. Cages, bedding, and foodwere autoclaved before use. Water was autoclaved, acidified to pH 2.8,and provided ad libitum.

Cell Inoculation

Mice were bilaterally ovariectomized (OVX) one week before tumor cellinoculation under anesthesia achieved by intraperitoneal injection of0.25 ml/animal of Avertin (amylic alcohol: 0.8 g/100 ml 0.9% NaCl; andtribromo ethanol: 2 g/100 ml 0.9% NaCl). 1.5×10⁶ ZR-75-1 cells inlogarithmic growth phase were harvested after the treatment of monolayerwith 0.05% trypsin/0.02% EDTA (w/v), were suspended in 0.1 ml of culturemedium containing 25% Matrigel and were inoculated subcutaneously onboth flanks of the animals using a 1 inch-long 20-gauge needle asdescribed previously (Dauvois et al., 1991). In order to facilitategrowth of the tumors, each animal received daily subcutaneous injectionof 10 μg of estradiol (E₂) in vehicle composed of 0.9% NaCl 5% ethanol1% gelatin for 5 weeks. After appearance of palpable ZR-75-1 tumors,tumor diameter was measured with calipers and mice having tumor diameterbetween 0.2 and 0.7 cm were selected for this study.

Hormonal Treatment

All animals, except those in the control OVX group, received dailysubcutaneous injections of 0.5 μg estrone (E₁) in 0.2 ml of 0.9% NaCl 5%ethanol 1% gelatin. In the indicated groups, DHEA was administeredpercutaneously twice daily at the doses of 0.3, 1.0 or 3.0 mg/animalapplied in a volume of 0.02 ml on the dorsal skin area outside the areaof tumor growth. DHEA was dissolved in 50% ethanol 50% propylene glycol.EM-800,((+)-7-pivaloyloxy-3-(4′-pivaloyloxyphenyl)-4-methyl-2-(4″-(2′″-piperidi-noethoxy)phenyl)-2H-benzopyran),was synthesized as described earlier (Gauthier et al., J. Med. Chem. 40:2117-2122, 1997) in the medicinal chemistry division of the Laboratoryof Molecular Endocrinology of the CHUL Research Center. EM-800 wasdissolved in 4% (v/v) ethanol 4% (v/v) polyethylene glycol (PEG) 600 1%(w/v) gelatin 0.9% (w/v) NaCl. Animals of the indicated groups receiveddaily oral doses of 15 μg, 50 μg, or 100 μg of EM-800 alone or incombination with DHEA while animals of the OVX group received thevehicle (0.2 ml 4% ethanol 4% PEG 600 1% gelatin 0.9% NaCl) alone.Tumors were measured once a week with Vernier calipers. Twoperpendicular diameters in cms (L and W) were recorded and tumor area(cm²) was calculated using the formula: L/2×W/2×π (Dauvois et al.,1991). The area measured on the first day of treatment was taken as 100%and changes in tumor size were expressed as percentage of initial tumorarea. In case of subcutaneous tumors in general, it is not possible toaccurately access three dimensional volume of tumor, therefore, onlytumors areas were measured. After 291 days (or 9.5 months) of treatment,the animals were sacrificed.

The categories of responses were evaluated as described (Dauvois et al.,1989a; Dauvois et al., 1989b; Labrie et al., 1995b). In short, partialregression corresponds to the tumors that regressed equal to or morethan 50% of their original size; stable response refers to tumors thatregressed less than 50% of the original size or progressed less than 50%of their original size, while complete regression refers to those tumorsthat were undetectable at the end of treatment. Progression refers totumors that progressed more than 50% compared with their original size.At the end of the experiment, all animals were killed by decapitation.Tumors, uterus, and vagina were immediately removed, freed fromconnective and adipose tissues, and weighed.

Statistical Analysis

Statistical significance of the effects of treatments on tumor size wasassessed using an analysis of variance (ANOVA) evaluating the effectsdue to DHEA, EM-800, and time, and repeated measures in the same animalsperformed at the initiation and at the end of the treatment (subjectswithin group factor). The repeated measures at time 0 and after 9.5months of treatment constitute randomized blocks of animals. The time isthus analyzed as a within-block effect while both treatments areassessed as between-block effects. All interactions between main effectswere included in the model. The significance of the treatment factorsand of their interactions was analyzed using the subjects within groupas the error term. Data were log-transformed. The hypotheses underlyingthe ANOVA assumed the normality of the residuals and the homogeneity ofvariance.

A posteriori pairwise comparisons were performed using Fisher's test forleast significant difference. Main effects and the interaction oftreatments on body weight and organ weight were analyzed using astandard two-way ANOVA with interactions. All ANOVAs were performedusing SAS program (SAS Institute, Cary, N.C., USA). Significance ofdifferences was declared using a 2-tailed test with an overall level of5%. Categorical data were analyzed with a Kruskall-Wallis test forordered categorical response variables (complete response, partialresponse, stable response, and progression of tumor). After overallassessment of a treatment effects, subsets of the results presented inTable 4 were analyzed adjusting the critical p-value for multiplecomparisons. The exact p-values were calculated using StatXact program(Cytel, Cambridge, Mass., USA). Data are expressed as means±standarderror of the mean (SEM) of 12 to 15 mice in each group.

Results

As illustrated in FIG. 3A, human ZR-75-1 tumors increased by 9.4-foldover 291 days (9.5 months) in ovariectomized nude mice treated with adaily 0.5 μg subcutaneously administered dose of estrone while incontrol OVX mice who received the vehicle alone, tumor size wasdecreased to 36.9% of the initial value during the course of the study.

Treatment with increasing doses of percutaneous DHEA caused aprogressive inhibition of E₁-stimulated ZR-75-1 tumor growth.Inhibitions of 50.4%, 76.8%, and 80.0% were achieved at 9.5 months oftreatment with the 0.3 mg, 1.0 mg, and 3.0 mg daily doses per animal ofDHEA, respectively (FIG. 3A). In agreement with the decrease in totaltumor load, treatment with DHEA led to a marked decrease of the averageweight of the tumors remaining at the end of the experiment. In fact,average tumor weight decreased from 1.12±0.26 g in controlE₁-supplemented ovariectomized nude mice to 0.37±0.12 g (P=0.005),0.20±0.06 g (P=0.001), and 0.17±0.06 g (P=0.0009) in the groups ofanimals receiving the daily 0.3, 1.0 and 3.0 mg doses of DHEA,respectively (FIG. 3B).

At the daily doses of 15 μg, 50 μg, and 100 μg, the antiestrogen EM-800inhibited estrogen-stimulated tumor size by 87.5% (P<0.0001), 93.5%(P<0.0001), and 94.0% (P=0.0003), respectively (FIG. 4A) when comparedto the tumor size in control animals at 9.5 months. The tumor sizereductions achieved with the three EM-800 doses are not significantlydifferent between each other. As illustrated in FIG. 3B, tumor weight atthe end of the 9.5-month study was decreased from 1.12±0.26 g in controlE₁-supplemented OVX mice to 0.08±0.03 g, 0.03±0.01 g and 0.04±0.03 g inanimals treated with the daily 15 μg, 50 μg, and 100 μg doses of EM-800,respectively (P<0.0001 at all doses of EM-800 vs E₁ supplemented OVX).

As mentioned above, the antiestrogen EM-800, at the daily oral dose of15 μg, caused a 87.5% inhibition of estrone-stimulated tumor growthmeasured at 9.5 months. The addition of DHEA at the three doses used hadno significant effect on the already marked inhibition of tumor sizeachieved with the 15 μg daily dose of the antiestrogen EM-800 (FIG. 4B).Thus, average tumor weight was dramatically reduced from 1.12±0.26 g incontrol estrone-supplemented mice to 0.08±0.03 g (P<0.0001), 0.11±0.04 g(P=0.0002), 0.13±0.07 g (P=0.0004) and 0.08±0.05 g (P<0.0001) in theanimals who received the daily dose of 15 μg of the antiestrogen aloneor in combination with the 0.3, 1.0, and 3.0 mg doses of DHEA,respectively (no significant difference was noted between the 4 groups)(FIG. 3B).

It was also of interest to examine the categories of responses achievedwith the above-indicated treatments. Thus, treatment with the increasingdoses of DHEA decreased, although not to a level of statisticalsignificance (P=0.088), the number of progressing tumors from 87.5% inthe control OVX animals supplemented with estrone to values of 50.0%,53.3%, and 66.7% in the animals treated with the daily doses of 0.3, 1.0or 3.0 mg of DHEA (Table 3). Complete responses, on the other hand,increased from 0% in the estrone-supplemented mice to 28.6%, 26.7%, and20.0% in the animals receiving the 0.3, 1.0, and 3.0 mg daily doses ofpercutaneous DHEA. Stable responses, on the other hand, were measured at12.5%, 21.4%, 20.0%, and 13.3% in the control E₁-supplemented mice andin the three groups of animals who received the above-indicated doses ofDHEA, respectively. In control ovariectomized mice, the rates ofcomplete, partial and stable responses were measured at 68.8%, 6.2%, and18.8%, respectively, while progression was seen in only 6.2% of tumors(Table 3).

Complete responses or disappearance of the tumors were achieved in29.4%, 33.3%, 26.7%, and 35.3% of tumors in the animals who received theantiestrogen EM-800 (P=0.0006) alone (15 μg) or in combination with the0.3 mg, 1.0 mg, or 3.0 mg of DHEA, respectively. Progression, on theother hand, was seen in 35.3%, 44.4%, 53.3%, and 17.6% of the tumors, inthe same groups of animals, respectively. There is no significantdifference between the groups treated with EM-800, either alone or incombination with DHEA.

No significant effect of DHEA or EM-800 treatment was observed on bodyweight adjusted for tumor weight. Treatment of OVX mice with estrone,increased uterine weight from 28±5 mg in OVX control mice to 132±8 mg(P<0.01) while increasing doses of DHEA caused a progressive butrelatively small inhibition of the stimulatory effect of estrone whichreached 26% (P=0.0008) at the highest dose of DHEA used. It can be seenin the same figure that estrone-stimulated uterine weight was decreasedfrom 132±8 mg in control estrone-supplemented mice to 49±3 mg, 36±2 mg,and 32±1 mg (P<0.0001 at all doses vs control) with the daily oral dosesof 15 μg, 50 μg, or 100 μg of EM-800 (overall P<0.0001), respectively.Fifteen micrograms (15 μg) EM-800 in combination with the 0.3 mg, 1.0 mgor 3.0 mg daily doses of DHEA, uterine weight was measured at 46±3 mg,59±5 mg and 69±3 mg, respectively.

On the other hand, treatment with estrone increased vaginal weight from14±2 mg in OVX animals to 31±2 mg (P<0.01) while the addition of DHEAhad no significant effect. Vaginal weight was then reduced to 23±1 mg,15±1 mg, and 11±1 mg following treatment with the daily 15 μg, 50 μg or100 μg doses of EM-800, respectively (overall p and pairwise P<0.0001 atall doses vs. control). In combination with the 0.3 mg, 1.0 mg or 3.0 mgdoses of DHEA and of EM-800, vaginal weight was measured at 22±1 mg,25±2 mg and 23±1 mg, respectively (N.S. for all groups versus 15 μgEM-800). It should be mentioned that at the highest dose used, namely100 μg daily, EM-800 decreased uterine weight in estrone-supplementedOVX animals to a value not different from that of OVX controls whilevaginal weight was reduced to a value below that measured in OVXcontrols (P<0.05). DHEA, probably due to its androgenic effects,partially counteracted the effect of EM-800 on uterine and vaginalweight.

TABLE 3 Effect of percutaneous administration of DHEA or oraladministration of EM-800 alone or in combination for 9.5 months on theresponses (complete, partial, stable, and progression) of human ZR-75-1breast tumor xenografts in nude mice. TOTAL CATEGORY OF RESPONSE NUMBEROF Complete Partial Stable Progression GROUP ANIMALS Number and (%) OVX16 11 (68.8)  1 (6.2) 3 (18.8) 1 (6.2)  OVX + E1 (0.5 μg) 16 0 (0)   0(0) 2 (12.5) 14 (87.5)  OVX + E1 (0.5 μg) + DHEA 0.3 mg 14 4 (28.6) 0(0) 3 (21.4) 7 (50.0) 1.0 mg 15 4 (26.7) 0 (0) 3 (20.0) 8 (53.3) 3.0 mg15 3 (20.0) 0 (0) 2 (13.3) 10 (66.7)  OVX + E1 (0.5 μg) + EM-800  15 μg17 5 (29.4) 1 (5.9) 5 (29.4) 6 (35.3)  50 μg 16 4 (25.0) 3 (18.8) 5(31.2) 4 (25.0) 100 μg  16 8 (50.0) 0 (0) 3 (18.8) 5 (31.2) OVX + E1(0.5 μg) + 0.3 mg 18 6 (33.3) 0 (0) 4 (22.2) 8 (44.4) EM-800 + DHEA 1.0mg 15 4 (26.7) 0 (0) 3 (20.0) 8 (53.3) 3.0 mg 17 6 (35.3) 0 (0) 8 (47.1)3 (17.6) E₁ = Estrone; DHEA = dehydroepiandrosterone; OVX =ovariectomized

Example 2 Example of Synthesis of the Preferred Compound of theInvention Synthesis of(S)-(+)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-1-benzopyranhydrochloride EM-01538 (EM-652, HCl)

Step A:

BF₃.Et₂O, toluene; 100° C.; 1 hour.

Step C:

3,4-dihydropyran, p-toluenesulfonic acid monohydrate, ethyl acetate; 25°C. under nitrogen, 16 hours, and then crystallization in isopropanol.

Steps D, E, and F:

(1) piperidine, toluene, Dean & Stark apparatus, reflux under nitrogen;(2) 1,8-diazabicyclo[5, 4, 0]undec-7-ene, DMF, reflux 3 hours;(3) CH₃MgCl, THF, −20 to 0° C. and then room temperature for 24 hours;

Steps G, H:

(1S)-(+)-10-camphorsulfonic acid, acetone, water, toluene, roomtemperature, 48 hours.

Step HH:

95% ethanol, 70° C., then room temperature 3 days.

Step HHR:

Recycling of mother liquor and wash of step HH (S)-10-camphorsulfonicacid, reflux; 36 hours, then room temperature for 16 hours.

Step I:

(1) DMF aq., Na₂CO₃, ethyl acetate;(2) Ethanol, dilute HCl;

(3) Water. Synthesis of2-tetrahydropyranyloxy-4-hydroxy-2′-(4″-tetrahydropyranyloxyphenyl)acetophenone (4)

A suspension of 2,4-dihydroxy-2′-(4″-hydroxyphenyl)acetophenone 3 (97.6g, 0.4 mole) (available from Chemsyn Science Laboratories, Lenexa,Kans.) in 3,4-dihydropyran (218 ml, 3.39 mole) and ethyl acetate (520ml) was treated with p-toluenesulfonic acid monohydrate (0.03 g, 0.158mmole) at about 25° C. The reaction mixture was stirred under nitrogenwith no external heating for about 16 hours. The mixture was then washedwith a solution of sodium bicarbonate (1 g) and sodium chloride (5 g) inwater (100 ml). The phases were separated and the organic phase waswashed with brine (20 ml). Each wash was back extracted with 50 ml ethylacetate. All the organic phases were combined and filtered throughsodium sulfate. Solvent (about 600 ml) was removed by distillation atatmospheric pressure and isopropanol (250 ml) was added. Additionalsolvent (about 300 ml) was distilled at atmospheric pressure andisopropanol (250 ml) was added. Additional solvent (about 275 ml) wasdistilled at atmospheric pressure and isopropanol (250 ml) was added.The solution was cooled at about 25° C. with stirring and after about 12hours, the crystalline solid was filtered, washed with isopropanol anddried (116.5 g, 70%).

Synthesis of4-hydroxy-4-methyl-2-(4′-[2″-piperidino]-ethoxy)phenyl-3-(4′″-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxy-chromane(10)

A solution of2-tetrahydropyranyloxy-4-hydroxy-2′-(4″-tetrahydropyranyloxyphenyl)acetophenone4 (1 kg, 2.42 mole), 4-[2-(1-piperidino)ethoxy]benzaldehyde 5 (594 g,2.55 mole) (available from Chemsyn Science Laboratories, Lenexa, Kans.)and piperidine (82.4 g, 0.97 mole) (available from Aldrich ChemicalCompany Inc., Milwaukee, Wis.) in toluene (8 L) was refluxed undernitrogen with a Dean & Stark apparatus until one equivalent of water (44mL) was collected. Toluene (6.5 L) was removed from the solution bydistillation at atmospheric pressure. Dimethylformamide (6.5 L) and1,8-diazabicyclo[5,4,0]undec-7-ene (110.5 g, 0.726 mole) were added. Thesolution was agitated for about 8 hours at room temperature to isomerizethe chalcone 8 to chromanone 9 and then added to a mixture of water andice (8 L) and toluene (4 L). The phases were separated and the toluenelayer washed with water (5 L). The combined aqueous washes wereextracted with toluene (3×4 L). The combined toluene extracts werefinally washed with brine (3×4 L), concentrated at atmospheric pressureto 5.5 L and then cooled to −10° C. With continued external cooling andstirring under nitrogen, a 3M solution of methylmagnesium chloride inTHF (2.5 L, 7.5 mole) (available from Aldrich Chemical Company Inc.,Milwaukee, Wis.) was added, maintaining the temperature below 0° C.After all the Grignard reagent was added, the external cooling wasremoved and the mixture allowed warm to room temperature. The mixturewas stirred at this temperature for about 24 hours. The mixture wasagain cooled to about −20° C. and with continued external cooling andstirring, saturated ammonium chloride solution (200 ml) was addedslowly, maintaining the temperature below 20° C. The mixture was stirredfor 2 hours and then added the saturated ammonium chloride solution (2L) and toluene (4 L) and agitated for five minutes. The phases wereseparated and the aqueous layer extracted with toluene (2×4 L). Thecombined toluene extracts were washed with dilute hydrochloric aciduntil the solution became homogenous and then with brine (3×4 L). Thetoluene solution was finally concentrated at atmospheric pressure to 2L. This solution was used directly in the next step.

Synthesis of(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (±12)

To the toluene solution of4-hydroxy-4-methyl-2-(4′-[-2″-piperidino]-ethoxy)-phenyl-3-(4′″-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxychromane(10) was added acetone (6 L), water (0.3 L) and (S)-10-camphorsulphonicacid (561 g, 2.42 mole) (available from Aldrich Chemical Company Inc.,Milwaukee, Wis.). The mixture was agitated under nitrogen for 48 hoursafter which time the solid(2R,S)-7-hydroxy-3-(4T-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (12) was filtered, washed withacetone and dried (883 g). This material was used in the next (HH) stepwithout further purification.

Synthesis of(2S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (13, (+)-EM-652(1S)-CSA salt)

A suspension of(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-benzopyran(1S)-10-camphorsulphonic acid salt ±12 (759 g) in 95% ethanol was heatedwith stirring to about 70° C. until the solid had dissolved. Thesolution was allowed to cool to room temperature with stirring thenseeded with a few crystals of(2S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt 13. The solution was stirred at roomtemperature for about three days in total. The crystals were filtered,washed with 95% ethanol and dried (291 g, 76%). The de of the productwas 94.2% and the purity 98.8%.

Synthesis of(S)-(+)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-1-benzopyranhydrochloride EM-01538 (EM-652, HCl)

A suspension of compound 13 (EM-652-(+)-CSA salt, 500 mg, 0.726 mmol) indimethylformamide (11 μL, 0.15 mmol) was treated with an 0.5 M aqueoussodium carbonate solution (7.0 mL, 3.6 mmol), and stirred for 15 min.The suspension was treated with ethyl acetate (7.0 mL) and stirredduring 4 h. The organic phase was then washed with an aqueous saturatedsodium carbonate solution (2×5 mL) and brine (1×5 mL) dried overmagnesium sulfate, and concentrated. A solution of the resulting pinkfoam (EM-652) in ethanol (2 mL) was treated with 2 N hydrochloric acid(400 μL, 0.80 mmol), stirred for 1 h, treated with distilled water (5mL), and stirred during 30 min. The resulting suspension was filtered,washed with distilled water (5 mL), dried in air and under high vacuum(65° C.) to give a creamy powder (276 mg, 77%): Fine off-white powder;Scanning calorimetry: Melting peak onset at 219° C., ΔH=83 J/g; [α]²⁴_(D)=154° in methanol 10 mg/ml.; ¹H NMR (300 MHz, CD₃OD) δ (ppm) 1.6(broad, 2H, H-4′″), 1.85 (broad, 4H, H-3″″ and 5″″), 2.03 (s, 3H, CH₃),3.0 and 3.45 (broad, 4H, H-2′″ and 6″″), 3.47 (t, J=4.9 Hz, 2H, H-3′″),4.26 (t, J=4.9 Hz, 2H, H-2′″), 5.82 (s, 1H, H-2), 6.10 (d, J=2.3 Hz, 1H,H-8), 6.35 (dd, J=8.4, 2.43 Hz, 1H, H-6), 6.70 (d, J=8.6 Hz, 2H, H-3′,and H-5′), 6.83 (d, J=8.7 Hz, 2H, H-3″ and H-5″), 7.01 (d, J=8.5 Hz, 2H,H-2′ and H-6′), 7.12 (d, J=8.4 Hz, 1H, H-5), 7.24 (d, J=8.6 Hz, 2H, H-2″and H-6″); ¹³C RMN (CD₃OD, 75 MHz) δ ppm 14.84, 22.50, 23.99, 54.78,57.03, 62.97, 81.22, 104.38, 109.11, 115.35, 116.01, 118.68, 125.78,126.33, 130.26, 130.72, 131.29, 131.59, 134.26, 154.42, 157.56, 158.96,159.33. Elemental Composition: C, H, N, Cl: Theory; 70.51, 6.53, 2.84,7.18, %, Found: 70.31, 6.75, 2.65, 6.89%.

Example 3 Materials and Methods Animals

Female BALB/c mice (BALB/cAnNCrlBR) weighing 18-20 g were obtained fromCharles-River, Inc. (St-Constant, Quebec, Canada) and housed 5 per cagein a temperature (23±1° C.)- and light (12 h light/day, lights on at7:15)-controlled environment. The mice were fed rodent chow and tapwater ad libitum. The animals were ovariectomized (OVX) under Isofluraneanesthesia via bilateral flank incisions and randomly assigned to groupsof 10 animals. Ten mice were kept intact as controls.

Treatments

In the first experiment (FIGS. 11 to 14), tested compounds, namelyEM-652.HCl, lasofoxifene (as free base; active and inactive enantiomers)and raloxifene, were administered orally by gavage once daily at dosesof 1, 3 or 10 μg/animal for 9 days, starting 2 days after ovariectomy.In the second experiment (Table 6), TSE 424 was administered orally bygavage once daily at doses of 1, 3, 10 or 30 μg/animal for 9 days,starting 2 days after ovariectomy. In both experiments, to evaluate theantiestrogenic activity, treatment with estrone (E₁, 0.0 μg, s.c.injection, twice daily) was started 5 days post-ovariectomy and wasadministered for a 6 day-period. Compounds were dissolved in ethanol (4%final concentration) and administered in 0.4% methylcellulose. Mice inthe intact and OVX control groups received the vehicle alone (4%ETOH-0.4% methylcellulose) during the 9-day period. The animals werekilled by exsanguination at the abdominal aorta on the 11th morningfollowing ovariectomy. The uteri and vagina were rapidly dissected,weighed, and kept in 10% buffered formalin for further histologicexamination.

Article I. Results Experiment 1:

As illustrated in FIG. 11, EM-652.HCl administered at the daily oraldoses of 1 μg, 3 μg, and 10 μg caused respective 24%, 48%, and 72%inhibitions of estrone-stimulated uterine weight (p<0.01 for all dosesversus control) while raloxifene administered at the same doses causedrespective 6% (NS), 14% (p<0.01) and 43% (p<0.01) inhibitions of thisparameter. Lasofoxifene (as free base), on the other hand, had noinhibitory effect at the lowest dose used while it caused respective 25%(p<0.01) and 44% (p<0.01) inhibitions of estrone-stimulated uterineweight at the daily doses of 3 μg and 10 μg. The inactive enantiomer oflasofoxifene exerted no inhibitory effect on this parameter at any doseused.

The compounds mentioned above exerted similar effects on vaginal weight.The daily oral administration of EM-652.HCl led to respective 10% (NS),25% and 53% inhibitions of vaginal weight (p<0.01 for the two highestdoses) at the 1 μg, 3 μg, and 10 μg doses (FIG. 12), while raloxifeneexerted a significant 24% (p<0.01) inhibitory effect on this parameterat the highest dose only (10 pz). Similarly to raloxifene, lasofoxifene(as free base) caused a significant 37% (p<0.01) inhibitory effect onlyat the highest dose used, while the inactive enantiomer had noinhibitory effect on vaginal weight at any dose used.

When compounds were administered alone (in the absence of estrone) toovariectomized mice at the daily oral doses of 1 μg and 10 μg,EM-652.HCl had no significant stimulatory effect on uterine weight atboth doses used, while treatment with 10 μg of lasofoxifene andraloxifene caused respective 93% (p<0.01) and 85% (p<0.01) stimulationsof uterine weight (FIG. 13), thus indicating an estrogenic effect ofthese latter compounds on this parameter. Similarly, EM-652.HCl exertedno significant stimulatory effect on vaginal weight (FIG. 14) whileadministration of 10 μg of lasofoxifene and raloxifene caused respective73% (p<0.01) and 56% (p<0.01) stimulations of vaginal weight. On theother hand, the inactive enantiomer of lasofoxifene had no stimulatoryeffect on uterine and vaginal weight.

Experiment 2:

As shown in table 4, TSE 424 administered at the daily oral doses of 1μg, 3 μg, 10 μg or 30 μg caused respective 12% (NS), 47%, 74%, and 94%inhibitions of estrone-stimulated uterine weight (p<0.01 for the threehighest doses versus E₁-control). On the other hand, the daily oraladministration of TSE 424 led to respective 16% (NS), 56% (p<0.01) and93% (p<0.01) inhibitions of vaginal weight at the 3 μg, 10 μg, and 30 μgdoses.

When the compound was administered alone (in the absence of estrone) toovariectomized mice at the daily oral doses of 3 μg and 30 μg, TSE 424had no significant stimulatory effect on uterine and vaginal weight atboth doses used (Table 4).

TABLE 4 Effect on uterine and vaginal weight of increasingconcentrations of TSE 424 administered orally for 9 days toovariectomized mice simultaneously treated or not with estrone. UTERINEWEIGHT VAGINAL WEIGHT TREATMENT (mg) (mg) INTACT  54.6 ± 12.5** 37.9 ±3.9** OVX  15.6 ± 1.3** 13.9 ± 1.5** OVX + E₁ 118.3 ± 6.0 53.4 ± 2.8OVX + E₁ + TSE 424 1 μg 105.5 ± 6.1 54.2 ± 3.0 OVX + E₁ + TSE 424 3 μg 69.7 ± 4.4** 47.2 ± 1.6 OVX + E₁ + TSE 424 10 μg  42.1 ± 2.7** 31.1 ±2.3** OVX + E₁ + TSE 424 30 μg  21.7 ± 1.7** 16.7 ± 1.8** OVX + TSE 4243 μg  18.3 ± 1.2 14.1 ± 1.2 OVX + TSE 424 30 μg  17.7 ± 1.6 15.3 ± 2.0**p < 0.01 versus E₁-treated control.

Example 4 Preventive Effects on Bone Loss, Serum Lipids and Total BodyFat. Animals and Treatment

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD(SD)Br)(Charles River Laboratory, St-Constant, Canada) weighing approximately220-270 g at start of treatment were used. The animals were acclimatizedto the environmental conditions (temperature: 22±3° C.; humidity:50±20%; 12-h light-12-h dark cycles, lights on at 07:15 h) for at least1 week before starting the experiments. The animals were housedindividually and were allowed free access to tap water and a pelletedcertified rodent feed (Lab Diet 5002, Ralston Purina, St-Louis, Mo.).Experiments were conducted in an animal facility approved by theCanadian Council on Animal Care (CCAC) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC) inaccordance with the CCAC Guide for Care and Use of Experimental Animals.

In a first experiment, one hundred fifty-four rats were randomlydistributed between 11 groups of 14 animals each as follows: 1) Intactcontrol; 2) OVX control; 3) OVX+E₂ (1 mg/kg); 4) OVX+EM-652.HCl (2.5mg/kg); 5) OVX+E₂+EM-652.HCl; 6) OVX+dehydroepiandrosterone (DHEA; 80mg/kg); 7) OVX+DHEA+EM-652.HCl; 8) OVX+DHEA+E₂; 9)OVX+DHEA+E₂+EM-652.HCl; 10) OVX+GW 5638; 11) OVX+E₂+GW 5638. On day 1 ofthe study, the animals of the appropriate groups were bilaterallyovariectomized (OVX) under isoflurane anesthesia. The DHEA was appliedtopically on the dorsal skin as a solution in 50% ethanol-50% propyleneglycol while the other tested compounds were administered as suspensionin 0.4% methylcellulose by oral gavage. Treatments were initiated on day2 of the study and were performed once daily during 3 months.

In the second experiment, one hundred thirty-two rats were randomlydistributed between 9 groups of 14 or 15 animals each as follows: 1)Intact control; 2) OVX control; 3) OVX+Premarin (0.25 mg/kg); 4)OVX+EM-652.HCl (2.5 mg/kg); 5) OVX+Premarin+EM-652.HCl; 6) OVX+TSE 424(2.5 mg/kg); 7) OVX+Premarin+TSE 424; 8) OVX+Lasofoxifene (tartratesalt; racemate; 2.5 mg/kg); 9) OVX+Premarin+Lasofoxifene. On day 1 ofthe study, the animals of the appropriate groups were bilaterally OVXunder isoflurane anesthesia. Tested compounds were administered assuspension in 0.4% methylcellulose by oral gavage. Treatments wereinitiated on day 2 of the study and were performed once daily during 26weeks. In both experiments, animals not receiving a test article weretreated with the appropriate vehicle alone during the same period.

Bone Mineral Density Measurements

After 3 months (experiment 1) or 26 weeks (experiment 2) of treatment,individual rats under Isoflurane anesthesia had their whole bodyskeleton and lumbar spine scanned using dual energy x-ray absorptiometry(DEXA; QDR 4500A, Hologic, Waltham, Mass.) and a Regional HighResolution Scan software. The bone mineral density (BMD) of the lumbarspine (vertebrae L2 to L4) and the total body composition (fatpercentage) were determined.

Serum Assays

After 3 months (experiment 1) or 26 weeks (experiment 2) of treatment,blood samples were collected at the jugular vein from overnight fastedanimals (under Isoflurane anesthesia). Samples were processed for serumpreparation and frozen at −80° C. until assay. Serum cholesterol levelsand alkaline phospatase activity (ALP) were determined using theBoehringer Mannheim Diagnostic Hitachi 911 Analyzer (Boehringer MannheimDiagnostic Laboratory Systems).

Statistical Analyses

Data are expressed as means±SEM. Statistical significance was determinedaccording to the multiple-range test of Duncan-Kramer (Kramer C Y;Biometrics 1956; 12:307-310).

Results

As shown in table 5, after 3 months of ovariectomy, BMD of the lumbarspine was 10% lower in OVX control animals than in intact controls(p<0.01). At the doses used, the administration of estradiol andEM-652.HCl alone prevented lumbar spine BMD loss by 98% (p<0.01) and 65%(p<0.05), respectively, while the combined treatment with E₂ andEM-652.HCl prevented the OVX-induced decrease in lumbar spine BMD by 61%(p<0.05). On the other hand, while the administration of DHEA aloneprevented lumbar spine BMD by 43% (p<0.05), the combined treatment withDHEA+E₂+EM-652.HCl prevented the OVX-induced decrease in lumbar spineBMD by 91% and led to BMD value not different from intact controls.

In table 6, 26 weeks after ovariectomy, BMD of the lumbar spine was 18%lowered compared to intact controls (p<0.01). The administration ofPremarin, EM-652.HCl, TSE 424 and Lasofoxifene alone prevented lumbarspine BMD by 54%, 62%, 49% and 61%, respectively (all p<0.01 versus OVXcontrols). The addition of Premarin to EM-652.HCl, TSE 424 orLasofoxifene led to lumbar spine BMD values not significantly differentfrom those obtained with the administration of each SERM alone (Table6). Similarly, the addition of DHEA to E₂ or to EM-652.HCl completelyprevented the OVX-induced decrease in lumbar spine BMD (Table 5). Thepositive effect of DHEA on BMD is also supported by its effect on serumalkaline phosphatase activity (ALP), a marker of bone formation andturnover. ALP activity was increased from 73±6 IU/L in OVX controlanimals to 224±18 IU/L, 290±27 IU/L, 123±8 IU/L and 261±20 IU/L (allp<0.01) in DHEA-, DHEA+EM-652.HCl-, DHEA+E₂- andDHEA+E₂+EM-652.HCl-treated animals, respectively, thus suggesting astimulatory effect of DHEA on bone formation (Table 7).

In addition to the preventive effects on bone loss, the administrationof EM-652.HCl, TSE 424, Lasofoxifene, GW 5638, DHEA and E₂ exerts somebeneficial effects on total body fat percentage and serum lipids. Afterthree months of ovariectomy, total body fat was increase by 22% (p<0.05;Table XXX 6). The administration of EM-652.HCl completely prevented theOVX-induced fat percentage increase while the addition of DHEA and/or E₂to the SERM led to fat percentage values below those observed in intactcontrol animals. After 26 weeks of ovariectomy, the 40% fat increaseinduced by estrogen deficiency was reversed by 74%, 78%, 75% and 114%following the administration of Premarin, EM-652.HCl, TSE 424 orLasofoxifene, respectively, while the addition of Premarin to each SERMcompletely prevented the OVX-induced fat percentage increase (Table 8).

As shown in Table 7, three months after ovariectomy, a 22% increase inserum cholesterol levels was observed in OVX control rats compared tointact controls (p<0.01). In fact, serum cholesterol was increased from2.01±0.11 mmol/L in intact animals to 2.46±0.08 mmol/L in OVX controls.The administration of E₂ or DHEA alone decrease serum cholesterol levelsto 1.37±0.18 mmol/L and 1.59±0.10 mmol/L, respectively, while theadministration of EM-652.HCl alone or in combination with E₂ and/or DHEAled to cholesterol levels significantly lower (between 0.65 to 0.96mmol/L) than those found in intact animals (2.01±0.11 mmol/L).Similarly, the administration of GW 5638, TSE 424 and lasofoxifene aloneor in combination with E₂ or Premarin completely prevented theOVX-induced increase on serum cholesterol levels and led to values lowerthan those found in intact animals (Tables 7 and 8).

TABLE 5 EFFECT ON PREVENTION OF BONE LOSS FOLLOWING 3 MONTH-TREATMENTWITH ESTRADIOL, EM-652•HCl, GW 5638 OR DHEA, ADMINISTERED ALONE OR INCOMBINATION, TO OVARIECTOMIZED FEMALE RATS ARTICLE III. LUMBAR SPINEPrevention ARTICLE II. BMD of Bone TREATMENT (g/cm²) Loss (%) 1) Intact0.2461 ± 0.0049** 100 OVX 0.2214 ± 0.0044 — OVX + E₂ 0.2457 ± 0.0049**98 OVX + EM-652•HCl 0.2374 ± 0.0027* 65 OVX + EM-652•HCl + E₂ 0.2364 ±0.0037* 61 OVX + DHEA 0.2321 ± 0.0034 43 OVX + DHEA + EM-652•HCl0.2458 + 0.0037** 99 OVX + DHEA + E₂ 0.2496 ± 0.0029** 114 OVX + DHEA +E₂ + EM- 0.2439 ± 0.0043** 91 652•HCl OVX + GW 5638 0.2299 ± 0.0060 34OVX + GW 5638 + E₂ 0.2344 ± 0.0054 53 *p < 0.05; **p < 0.01,experimental versus OVX control rats.

TABLE 6 EFFECT ON PREVENTION OF BONE LOSS FOLLOWING 26 WEEK- TREATMENTWITH PREMARIN, EM-652•HCl, TSE 424 OR LASOFOXIFENE, ADMINISTERED ALONEOR IN COMBINATION WITH PREMARIN, TO OVARIECTOMIZED FEMALE RATS ARTICLEV. LUMBAR SPINE ARTICLE IV. Prevention ARTICLE VI. BMD of Bone TREATMENT(g/cm²) Loss (%) 1) Intact 0.2482 ± 0.0067** 100 OVX 0.2035 ± 0.0035 —OVX + Premarin 0.2277 ± 0.0028** 54 OVX + EM-652•HCl 0.2311 ± 0.0040**62 OVX + Premarin + EM-652•HCl 0.2319 + 0.0057** 64 OVX + TSE 424 0.2252± 0.0058** 49 OVX + Premarin + TSE 424 0.2223 ± 0.0046** 42 OVX +Lasofoxifene 0.2307 ± 0.0040** 61 OVX + Premarin + Lasofoxifene 0.2357 ±0.0035** 72 **p < 0.01, experimental versus OVX control rats.

TABLE 7 EFFECT ON TOTAL BODY FAT PERCENTAGE, SERUM CHOLESTEROL LEVELSAND ALKALINE PHOSPHATASE ACTIVITY FOLLOWING 3 MONTH-TREATMENT WITHESTRADIOL, EM-652•HCl, GW 5638 OR DHEA, ADMINISTERED ALONE OR INCOMBINATION, TO OVARIECTOMIZED FEMALE RATS ARTICLE VIII. ARTICLE IX.ARTICLE X. ARTICLE VII. TOTAL FAT CHOLESTEROL ALP ARTICLE XI. TREATMENT(%) (mmol/L) (IU/L) 1) Intact 24.0 ± 1.5* 2.01 ± 0.11**  39 ± 2** OVX29.2 ± 1.5 2.46 ± 0.08  73 ± 6 OVX + E₂ 19.5 ± 2.5** 1.37 ± 0.18**  59 ±4 OVX + EM-652•HCl 23.2 ± 1.4** 0.87 ± 0.04**  91 ± 6* OVX +EM-652•HCl + E₂ 20.4 ± 1.4** 0.96 ± 0.07**  92 ± 5* OVX + DHEA 17.3 ±1.5** 1.59 ± 0.10** 224 ± 18** OVX + DHEA + EM-652•HCl 18.0 ± 1.1** 0.65± 0.06** 290 ± 27** OVX + DHEA + E₂ 15.8 ± 1.3** 1.08 ± 0.08** 123 ± 8**OVX + DHEA + E₂ + EM- 19.2 ± 1.6** 0.71 ± 0.08** 261 ± 20** 652•HClOVX + GW 5638 21.9 ± 1.4** 1.14 ± 0.08**  72 ± 6 OVX + GW 5638 + E₂ 23.2± 1.2** 0.91 ± 0.07**  80 ± 6 *p < 0.05; **p < 0.01, experimental versusOVX control rats.

TABLE 8 EFFECT ON TOTAL BODY FAT PERCENTAGE, SERUM CHOLESTEROL LEVELSAND ALKALINE PHOSPHATASE ACTIVITY FOLLOWING 26 WEEK-TREATMENT WITHPREMARIN, EM-652•HCl, TSE 424 OR LASOFOXIFENE, ADMINISTERED ALONE OR INCOMBINATION WITH PREMARIN, TO OVARIECTOMIZED FEMALE RATS ARTICLE XIII.ARTICLE XIV. ARTICLE XV. ARTICLE XII. TOTAL FAT CHOLESTEROL ALP ARTICLEXVI. TREATMENT (%) (mmol/L) (IU) 1) Intact 25.5 ± 1.8** 2.11 ± 0.11** 33 ± 2* OVX 35.7 ± 1.6 2.51 ± 0.09  60 ± 6 OVX + Premarin 28.2 ± 1.8**1.22 ± 0.07**  49 ± 3 OVX + EM-652•HCl 27.7 ± 1.4** 0.98 ± 0.06**  78 ±4 OVX + EM-652•HCl + 25.7 ± 2.2** 1.10 ± 0.07**  81 ± 6 Premarin OVX +TSE 424 28.0 ± 1.8** 1.15 ± 0.05**  85 ± 6 OVX + TSE 424 + Premarin 25.7± 1.7** 1.26 ± 0.14**  98 ± 22** OVX + Lasofoxifene 24.1 ± 1.3** 0.60 ±0.02** 116 ± 9** OVX + Lasofoxifene + Premarin 23.8 + 1.9** 0.81 ±0.12** 107 ± 6** *p < 0.05; **p < 0.01, experimental versus OVX controlrats.

Example 5

Preventive Effects on Bone Loss Following Treatment with the SermsEM-652.HCl, TSE-424 and ERA-923, Administered Alone and in Combinationwith DHEA to Ovariectomized Female Rats

Animals and Treatment

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD(SD)Br)(Charles River Laboratory, St-Constant, Canada) weighing approximately220-270 g at start of treatment were used. The animals were acclimatizedto the environmental conditions (temperature: 22±3° C.; humidity:50±20%; 12-h light-12-h dark cycles, lights on at 07:15 h) for at least1 week before starting the experiments. The animals were housedindividually and were allowed free access to tap water and a pelletedcertified rodent feed (Lab Diet 5002, Ralston Purina, St-Louis, Mo.).Experiments were conducted in an animal facility approved by theCanadian Council on Animal Care (CCAC) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC) inaccordance with the CCAC Guide for Care and Use of Experimental Animals.

One hundred twenty-six rats were randomly distributed between 9 groupsof 14 animals each as follows: 1) Intact control; 2) OVX control; 3)OVX+EM-652.HCl (2.5 mg/kg); 4) OVX+TSE-424 (EM-4803, 2.5 mg/kg); 5)OVX+ERA-923 (EM-3527, 2.5 mg/kg); 6) OVX+dehydroepiandrosterone (DHEA;80 mg/kg); 7) OVX+DHEA+EM-652.HCl; 8) OVX+DHEA+TSE-424; 9)OVX+DHEA+ERA-923. On day 1 of the study, the animals of the appropriategroups were bilaterally ovariectomized (Ovx) under isofluraneanesthesia. The DHEA was applied topically on the dorsal skin as asolution in 50% ethanol-50% propylene glycol while the tested SERMs wereadministered as suspension in 0.4% methylcellulose by oral gavage.Treatments were initiated on day 2 of the study and were performed oncedaily during 5 weeks.

Bone Mineral Density Measurements

After 5 weeks of treatment, individual rats under Isoflurane anesthesiahad their lumbar spine, femur and tibia scanned using dual energy x-rayabsorptiometry (DEXA; QDR 4500A, Hologic, Waltham, Mass.) and a RegionalHigh Resolution Scan software. The bone mineral density (BMD) of thelumbar spine (vertebrae L2 to L4), distal femoral metaphysis (DFM) andproximal tibial metaphysis (PTM) were determined.

Statistical Analyses

Data are expressed as means±SEM. Statistical significance was determinedaccording to the multiple-range test of Duncan-Kramer (Kramer C Y 1956).

Results

As shown in table 9, after 5 weeks of ovariectomy, BMD of the lumbarspine was 9% lower in Ovx control animals than in intact controls. Atthe dose used the administration of the SERMs: EM-652.HCl, TSE-424 orERA-923 alone prevented lumbar spine BMD loss by 86%, 53% and 78%,respectively. On the other hand, the administration of DHEA aloneprevented lumbar spine BMD loss by 44%, while the combined treatmentwith DHEA+EM-652.HCl, DHEA+TSE-424 or DHEA+ERA-923 prevented theOVX-induced decrease in lumbar spine BMD by 94%, 105% and 105%,respectively.

Bone mineral density of the distal femoral metaphysis (DFM) wasdecreased by 10% after 5 weeks of ovariectomy (Table 9). Theadministration of the SERMs: EM-652.HCl, TSE-424 or ERA-923 aloneprevented DFM BMD loss by 95%, 70% and 83%, respectively. On the otherhand, the administration of DHEA alone prevented DFM BMD loss by 71%,while the combined treatment with DHEA+EM-652.HCl, DHEA+TSE-424 orDHEA+ERA-923 completely prevented the OVX-induced decrease in DFM BMDand led to DFM BMD values higher than those observed in intact controlanimals. Similar results were obtained on proximal tibial metaphysis BMD(Table 9).

TABLE 9 EFFECT ON PREVENTION OF BONE LOSS FOLLOWING 5 WEEK-TREATMENTWITH THE SERMs EM-652.HCl, TSE-424 AND ERA-923, ADMINISTERED ALONE OR INCOMBINATION WITH DHEA, TO OVARIECTOMIZED FEMALE RATS DISTAL FEMORALPROXIMAL TIBIAL LUMBAR SPINE METAPHYSIS (DFM) METAPHYSIS (PFM) (L2-L4)Prevention Prevention BMD Prevention of BMD of Bone BMD of Bone LossTREATMENT (g/cm²) Bone Loss (%) (g/cm²) Loss (%) (g/cm²) (%) Intact0.2261 ± 0.0046 100 0.3024 ± 0.0040 100 0.2828 ± 0.0032 100 Ovx 0.2051 ±0.0037 — 0.2709 ± 0.0036 — 0.2560 ± 0.0028 — Ovx + EM-652.HCl 0.2232 ±0.0031 86 0.3008 ± 0.0055 95 0.2806 ± 0.0035 92 Ovx + TSE-424 0.2162 ±0.0035 53 0.2929 ± 0.0042 70 0.2750 ± 0.0039 71 Ovx + ERA-923 0.2214 ±0.0029 78 0.2969 ± 0.0029 83 0.2805 ± 0.0034 91 Ovx + DHEA 0.2144 ±0.0028 44 0.2934 ± 0.0046 71 0.2672 ± 0.0041 42 Ovx + DHEA + EM-652.HCl0.2249 ± 0.0023 94 0.3122 ± 0.0045 131 0.2867 ± 0.0047 115 Ovx + DHEA +TSE-424 0.2271 ± 0.0030 105 0.3099 ± 0.0040 124 0.2833 ± 0.0034 102Ovx + DHEA + ERA-923 0.2271 ± 0.0030 105 0.3072 ± 0.0053 115 0.2817 ±0.0034 96

Example 6 Effect of Compounds of the Invention on Alkaline PhosphataseActivity in Human Endometrial Adenocarcinoma Ishikawa Cells. MaterialsMaintenance of Stock Cell Cultures

The human Ishikawa cell line derived from a well differentiatedendometrial adenocarcinoma was kindly provided by Dr. Erlio Gurpide, TheMount Sinai Medical Center, New York, N.Y. The Ishikawa cells wereroutinely maintained in Eagle's Minimum Essential Medium (MEM)containing 5% (vol/vol) FBS (Fetal Bovine Serum) and supplemented with100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential aminoacids solution. Cells were plated in Falcon T75 flasks at a density of1.5×10⁶ cells at 37° C.

Cell Culture Experiments

Twenty four hours before the start of the experiment, the medium of nearconfluent Ishikawa cells was replaced by fresh estrogen-free basalmedium (EFBM) consisting of a 1:1 (v:v) mixture of phenol red-free Ham'sF-12 and Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 100U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, and 5% FBStreated twice with dextran-coated charcoal to remove endogenoussteroids. Cells were then harvested by 0.1% pancreatin (Sigma) and 0.25mM HEPES, resuspended in EFBM and plated in Falcon 96, wellflat-bottomed microtiter plates at a density of 2.2×10⁴ cells/well in avolume of 100 μl and allowed to adhere to the surface of the plates for24 h. Thereafter, medium was replaced with fresh EFBM containing theindicated concentrations of compounds in a final volume of 200 μl. Cellswere incubated for five days, with a medium change after 48 h.

Alkaline Phosphatase Assay

At the end of the incubation period, microtiter plates were inverted andgrowth medium was decanted. The plates were rinsed with 200 μl by wellof PBS (0.15M NaCl, 10 mM sodium phosphate, pH 7.4). PBS was thenremoved from the plates while carefully leaving some residual PBS, andthe wash procedure was repeated once. The buffered saline was thendecanted, and the inverted plates were blotted gently on a paper towel.Following replacement of the covers, the plates were placed at −80° C.for 15 min followed by thawing at room temperature for 10 min. Theplates were then placed on ice, and 50 μl of an ice-cold solutioncontaining 5 mM p-nitrophenyl phosphate, 0.24 mM MgCl₂, and 1 Mdiethanolamine (pH 9.8) were added. Plates were then warmed to roomtemperature, and the yellow color from the production of p-nitrophenylwas allowed to develop (8 min). Plates were monitored at 405 nm in anenzyme-linked immunosorbent assay plate reader (BIO-RAD, model 2550 EIAReader).

Calculations

Dose-response curves as well as IC₅₀ values were calculated using aweighted iterative nonlinear squares regression.

TABLE 10 Inhibition of Maximal 1 nM E₂- Maximal stimulation of inducedinhibition of alkaline stimulation of 1 nM E₂-induced phosphatasealkaline stimulation of % of 1 nM E₂ phosphatase alkaline stimulation *IC₅₀ (nM) phosphatase (nb of (nb of (nb of NAME CODE NAME STRUCTUREexperiments) experiments) experiments) EM-652•HCl (Acolbifene)EM-652•HCl; (EM-1538)

1.88 ± 0.26 (22) 1.52 ± 0.22 (18) 98.97 ± 0.174 (18) OH-ToremifeneEM-880 

29.6 ± 2.1  (6) 72.1 ± 7.6  (3) 75.73 ± 3.52  (3) GW-5638 EM-1796

7.75 ± 5.5  (2) No inhibition Raloxifene LY 156758 EM-1105

12.8 ± 1.7  (8) 3.39 ± 0.9  (6) 94.31 ± 1.74  (5) LY 353381 EM-1665

15.5 ± 0.25 (5) 1.87 ± 0.07 (2) 90.25 ± 0.127 (2) Lasofoxifene (freebase) EM-3114

17.9 (1) 4.24 (1)  85.14 (1) ERA-923 EM-3527

 0.6 (1) 5.84 (1) 100.16 (1) * % of 1 nM E₂ stimulation = OD 405 nmcompound-OD 405 nm basal/OD 405 nm 1 nM E₂-OD 405 nm basal Please seealso Labrie et al. 1999.

Example 7 Effect of EM-652.HCl, TSE 424, and Lasofoxifene on theProliferation of Human Breast Cancer MCF-7 Cells Methods: Maintenance ofStock Cell Cultures

MCF-7 human breast cancer cells were obtained from the American TypeCulture Collection # HTB 22 at passage 147 and routinely grown in phenolred-free Dulbecco's Modified Eagle's-Ham's F12 medium, the supplementsmentioned above and 5% FBS. The MCF-7 human breast adenocarcinoma cellline was derived from the pleural effusion of a Caucasian 69-year-oldfemale patient. MCF-7 cells were used between passages 148 and 165 andsubcultured weekly

Cell Proliferation Studies

Cells in their late logarithmic growth phase were harvested with 0.1%pancreatin (Sigma) and resuspended in the appropriate medium containing50 ng bovine insulin/ml and 5% (v/v) FBS treated twice withdextran-coated charcoal to remove endogenous steroids. Cells were platedin 24-well Falcon plastic culture plates (2 cm²/well) at the indicateddensity and allowed to adhere to the surface of the plates for 72 h.Thereafter, medium was replaced with fresh medium containing theindicated concentrations of compounds diluted from 1000×stock solutionsin 99% redistilled ethanol in the presence or absence of E₂. Controlcells received only the ethanolic vehicle (0.1% EtOH, v/v). Cells wereincubated for the specified time intervals with medium changes at 2- or3-day intervals. Cell number was determined by measurement of DNAcontent.

Calculations and Statistical Analysis

Dose-response curves as well IC₅₀ values were calculated using aweighted iterative nonlinear least-squares regression. All results areexpressed as means±SEM.

TABLE 11 Experiment 1 Maximal stimulation of DNA by tested Inhibition of1 nM E₂ compounds stimulation of DNA % of 1 nM E₂ by tested compoundsNAME CODE NAME stimulation * IC₅₀ (nM) EM-652•HCl EM-652•HCl; N.S. 0.796EM-1538 TSE 424 EM-3527 N.S. 3.68 Experiment 2 Stimulation of DNA bytested Inhibition of 1 nM E₂ compounds stimulation of DNA % of 1 nM E₂by tested compounds NAME CODE NAME stimulation * IC₅₀ (nM) EM-652•HClEM-652•HCl; N.S. 0.205 EM-1538 Lasofoxifene EM-3114 N.S. 0.379 (freebase)

Example 8 Comparison of the Effects of EM-652.Hcl, Tamoxifen,Toremifene, Droloxifene, Idoxifene, GW-5638, and Raloxifene on theGrowth of Human RZ-75-1 Breast Tumors in Nude Mice.

The objective of this example was to compare the agonistic andantagonistic effects of EM-652.HCl and six other oral antiestrogens(SERMs) on the growth of the well-characterized estrogen-sensitiveZR-75-1 breast cancer xenografts in ovariectomized nude mice.

Materials and Methods Human ZR-75-1 Breast Cancer Cells

ZR-75-1 human breast cancer cells were obtained from the American TypeCulture Collection (Rockville, Md.) and cultured in phenol red-freeRPMI-1640 medium. The cells were supplemented with 2 mM L-glutamine, 1mM sodium pyruvate, 100 IU penicillin/ml, 100 μg streptomycin/ml, and10% (v/v) fetal bovine serum and incubated under an humidifiedatmosphere of 95% air/5% CO2 at 37° C. Cells were passaged weekly andharvested at 85-90% confluence using 0.083% pancreatin/0.3 mM EDTA.

Animals and Tumor Inoculation

Homozygous female nu/nu Br athymic mice (28- to 42-day old) wereobtained from Charles River, Inc. (Saint-Constant, Québec, Canada). Themice (5 per cage) were housed in vinyl cages equipped with air filterlids, which were kept in laminar airflow hoods and maintained underpathogen-limiting conditions. The photoperiod was 12 hours of light and12 hours of darkness (lights on at 07:15). Cages, bedding and food(Agway Pro-Lab R-M-H Diet #4018) were autoclaved before use. Water wasautoclaved and provided ad libitum. Bilateral ovariectomy was performedunder isoflurane-induced anesthesia. At the time of ovariectomy, animplant of estradiol (E₂) was inserted subcutaneously to stimulateinitial tumor growth. E₂ implants were prepared in 1 cm-long Silastictubing (inside diameter: 0.062 inch; outside diameter: 0.095 inch)containing 0.5 cm of a 1:10 (w/w) mixture of estradiol and cholesterol.One week after ovariectomy, 2×10 6 ZR-75-1 (passage 93) cells wereinoculated subcutaneously in 0.1 ml of RPMI-1640 medium+30% Matrigel onboth flanks of each ovariectomized (OVX) mouse through a 2.5-cm-long22-gauge needle. After four weeks, the E₂ implants were replaced in allanimals by estrone-containing implants of the same size (E1:chol, 1:25,w:w). Randomization and treatments were started one week later.

Treatments

One day prior to initiation of treatments, 255 mice bearing ZR-75-1tumors of an average area of 24.4±0.4 mm2 (range 5.7 to 50.7 mm²) wererandomly assigned to 17 groups (with respect to tumor size), eachcontaining 15 mice (total of 29 or 30 tumors). The 17 groups includedtwo control groups (OVX and OVX+Estrone), seven groups supplemented withan estrone implant and treated with an antiestrogen and eight othergroups that received an antiestrogen alone. The estrone implants werethen removed from the animals in the ovariectomized control group (OVX)and in groups that were to receive the antiestrogen alone.Estrone-containing implants in the nine other groups were changedthereafter every 6 weeks. EM-652.HCl, raloxifene, droloxifene, idoxifeneand GW 5638 were synthesized in the medicinal chemistry division of theOncology and Molecular Endocrinology Research Center. Tamoxifen waspurchased from Plantex (Netanya, Israël) while toremifene citrate waspurchased from Orion (Espoo, Finland). Under estrone stimulation, theantiestrogens were given at the daily oral dose of 50 μg (2 mg/kg, onaverage) suspended in 0.2 ml of 0.4% (w/v) methylcellulose. In theabsence of estrone stimulation, animals were treated with 200 μg (8mg/kg on average) of each antiestrogen once daily by the oral route.Animals in both control groups received 0.2 ml of the vehicle alone. Theantiestrogen suspensions at the appropriate concentration were preparedeach month, stored at 4° C. and used under constant agitation. Powderstock were hermetically stored at 4° C. (idoxifene, raloxifene,toremifene, GW 5638, droloxifene) or at room temperature (tamoxifen,EM-652.HCl).

Tumor Measurements and Necropsy

Two perpendicular diameters were recorded and tumor area (mm2) wascalculated using the formula: L/2×W/2×π. The area measured on the firstday of treatment was taken as 100%.

After 161 days of treatment, the remaining animals were anesthetizedwith isoflurane and killed by exsanguination. To further characterizethe effect of the estrogen and antiestrogens, estrogen-responsivetissues, such as the uterus and vagina, were immediately removed, freedfrom connective and adipose tissue and weighed. The uteri were preparedto evaluate endometrial thickness by image analysis performed with ImagePro-Plus (Media Cybernetics, Maryland, USA). In brief, uteri were fixedin 10% formalin and embedded in parafin. Hematoxylin- and eosin-stainedsections of mice uteri were analyzed. Four images per uterus (2 peruterine horn) were analyzed. Mean epithelial cell height was measured inall animals of each group.

Response Criteria

Tumor response was assessed at the end of the study or at death of eachanimal, if it occurred during the course of the experiment. In thiscase, only data of mice that survived for at least half of the study (84days) were used in the tumor response analysis. In brief, completeregression identifies those tumors that were undetectable at the end ofthe experiment; partial regression corresponds to the tumors thatregressed ≥50% of their original size; stable response refers to tumorsthat regressed <50% or progressed 50%; and progression refers to tumorsthat progressed ≥50% compared with their original size.

Statistical Analyses

The change in total tumors surface areas between day 1 and day 161 wereanalyzed according to an ANOVA for repeated measurements. The modelincluded the treatment, time, and time-treatment interaction effectsplus the term to account for the strata at randomization. Thesignificance of the different treatments effects at 161 days was thustested by the time-treatment interaction. Analysis of the residualsindicated that the measurements on the original scale were not fittedfor analysis by an ANOVA nor any of the transformations that were tried.The ranks were therefore selected for the analyses. The effect of thetreatments on the epithelial thickness was assessed by a one-way ANOVAincluding also the strata at randomization. A posteriori pairwisecomparisons were performed using least square means statistics. Theovervall type 1 error rate (a) was controlled at 5% to declaresignificance of the differences. All calculations were performed usingProc MIXED on the SAS Software (SAS Institute, Carry, N.C.).

Results Antagonistic Effects on ZR-75-1 Tumor Growth

Estrone alone (OVX+E₁) caused a 707% increase in ZR-75-1 tumor sizeduring the 23 week-treatment period (FIG. 18). Administration of thepure antiestrogen EM-652.HCl at the daily oral dose of 50 μg toestrone-stimulated mice completely prevented tumor growth. In fact, notonly tumor growth was prevented but after 23 weeks of treatment, tumorsize was 26% lower than the initial value at start of treatment(p<0.04). This value obtained after treatment with EM-652.HCl was notstatistically different from that observed after ovariectomy alone (OVX)where tumor size decreased by 61% below initial tumor size. At the samedose (50 μg) and treatment period, the six other antiestrogens did notdecrease initial average tumor size. Tumors in these groups were allsignificantly higher than the OVX control group and to theEM-652.HCl-treated group (p<0.01). In fact, compared to pretreatmentvalues, 23 weeks of treatment with droloxifene, toremifene, GW 5638,raloxifene, tamoxifen and idoxifene led to average tumor sizes 478%,230%, 227%, 191%, 87% and 86% above pretreatment values, respectively(FIG. 18).

Agonistic Effects on ZR-75-1 Tumor Growth

After 161 days of treatment with a daily dose of 200 μg of tamoxifen, inthe absence of estrone supplementation, the average tumor size increasedto 196% over baseline (p<0.01 vs OVX) (FIG. 19). On the other hand, theaverage tumor size of mice treated with Idoxifene increased (125%)(p<0.01) while tumor size in mice treated with toremifene increased by86% (p<0.01) (FIG. 19). The addition of 200 μg of EM-652.HCl to 200 μgof tamoxifen completely inhibited the proliferation observed withtamoxifen alone (FIG. 20). On the other hand, treatment with EM-652.HCl(p=0.44), raloxifene (p=0.11), droloxifene (p=0.36) or GW 5638 (p=0.17)alone did not significantly change ZR-75-1 tumor size compared to theOVX control group, at the end of the experiment. (FIG. 19).

Effects on Categories Response

Effects of 50 μg of antiestrogen on estrone stimulation. In addition tothe effect on tumor size, the category of response achieved by eachindividual tumor at the end of the experiment is an important parameterof treatment efficacy. In ovariectomized mice, complete, partial, andstable responses were achieved in 21%, 43% and 38% of tumors,respectively, and none of the tumors progressed. On the other hand, inOVX animals supplemented with estrone, 100% of tumors have progressed(FIG. 21). In the EM-652.HCl-treated group of OVX animals supplementedwith estrone, complete, partial, and stable responses were seen in 17%,17%, and 60% of tumors, respectively and only 7% (2 tumors out of 30)have progressed. Under the same conditions of estrone stimulation,treatment with a daily 50 μg dose of any of the other antiestrogens wasunable to decrease the percentage of progressing tumors under 60%. Infact, 65% of tumors (17 of 26) progressed in the tamoxifen-treatedgroup, while 89% (25 of 28) progressed with toremifene, 81% progressed(21 of 26) with raloxifene, 100% (23 of 23) progressed with droloxifene,while 71% (20 of 28) progressed with idoxifene and 77% (20 of 26)progressed with GW 5638 (FIG. 21).

Effects of 200 μg of Antiestrogen in the Absence of Estrone Stimulationon Categories Response

As illustrated in FIG. 22, tamoxifen, idoxifene and toremifene led togreater proportion of progressing tumors, in the absence of estronestimulation, than the other antiestrogens. In fact, 62% (16 of 26), 33%(8 of 24) and 21% (6 of 28) of tumors were in the progression categoryafter tamoxifen-, idoxifene- and toremifene treatment at the daily doseof 200 μg, respectively. As can be seen in FIG. 23, the addition of 200μg of EM-652.HCl to tamoxifen reduced the percentage of progressingtumors with tamoxifen alone from 62% (16 of 26) to 7% when EM-652.HClwas added to tamoxifen (2 of 28).

Effects of Antiestrogens on Thickness of Uterine Epithelial Cells

The height of the endometrial epithelial cells was measured as the mostdirect parameter of agonistic and antagonistic effect of each compoundin the endometrium.

Effect of Daily 50 μg of Antiestrogen in the Presence of EstroneStimulation on Thickness of Uterine Epithelial Cells

At the daily oral dose of 50 μg, EM-652.HCl inhibited the stimulatoryeffect of estrone on epithelial height by 70%. The efficacy of the sixother antiestrogens tested were significantly lower (p<0.01). In fact,droloxifene, GW 5638, raloxifene, tamoxifen, toremifene and idoxifeneinhibited estrone stimulation by 17%, 24%, 26%, 32%, 41% and 50%,respectively. (Table 12).

Effect of Daily 200 μg of Antiestrogen in Absence of Estrone Stimulationon Thickness of Uterine Epithelial Cells

In the absence of estrone stimulation, EM-652 HCl and droloxifene werethe only compounds tested that did not significantly increase the heightof epithelial cells (114% and 101% of the OVX control group value,respectively). Tamoxifen (155%), toremifene (135%) and idoxifene (176%)exerted a significant stimulation of uterine epithelial height (p<0.01vs OVX control group). Raloxifene (122%) and GW 5638 (121%) also exerteda statistically significant stimulation of uterine epithelial height(p<0.05 vs OVX control group (Table 12). The agonistic and antagonisticeffects of each antiestrogen measured on uterine and vaginal weight werein accordance with the pattern observed on uterine epithelium thickness(Data not shown).

TABLE 12 ENDOMETRIAL EPITHELIUM THICKNESS GROUP n (μm) ± SEM OVX CONTROL14   18.31 ± 0.04 OVX + E₁ CONTROL 8 40.58^(b,d) ± 0.63 OVX + E₁ +EM-652•HCl 14   25.06^(b) ± 0.07 OVX + E₁ + TAMOXIFEN 10 33.44^(b,d) ±0.04 OVX + E₁ + TOREMIFENE 13 31.47^(b,d) ± 0.04 OVX + E₁ + RALOXIFENE12 34.72^(b,d) ± 0.06 OVX + E₁ + DROLOXIFENE 12 36.71^(b,d) ± 0.12 OVX +E₁ + IDOXIFENE 12 29.35^(b,d) ± 0.05 OVX + E₁ + GW 5638 12 35.30^(b,d) ±0.07 OVX + EM-652•HCl 12   20.79 ± 0.10 OVX + TAMOXIFEN 11 28.47^(b,d) ±0.05 OVX + EM-652•HCl + 13 27.95^(b,d) ± 0.06 TAMOXIFEN OVX + TOREMIFENE13 24.75^(b,c) ± 0.04 OVX + RALOXIFENE 12   22.33^(a) ± 0.05 OVX +DROLOXIFENE 13   18.50 ± 0.07 OVX + IDOXIFENE 11 32.14^(b,d) ± 0.05OVX + GW 5638 13   22.22^(a) ± 0.05 ^(a,b)Experimental versus OVXcontrol mice: ^(a)P < 0.05; ^(b)P < 0.01. ^(c,d)Experimental versusEM-652•HCl treated-mice: ^(c)P < 0.05; ^(d)P < 0.01.

Example 9

Radioactivity in the Brain of Female Rats Following a Single Oral Doseof ¹⁴C-EM-800 (20 mg/kg)

Example 8 shows the radioactivity in brain of rats following single oraldose of ¹⁴C-EM-800 (20 mg/kg), a SERM of the present invention. Forcomparison purposes, values for the blood, plasma, liver (Table 13) anduterus from each of these animals were included. Tissue Distribution andExcretion of Radioactivity Following a Single Oral Dose of ¹⁴C-EM-800(20 mg/2 ml/kg) to Male and Female Long-Evans Rats. These numbersindicate that the amount of total drug-derived radioactivity in thebrain of female Long-Evans rats was very low (ng equiv/g tissue) and wasnot detected after 12 hr post dose. At 2 hours, radioactivity in thebrain was 412 lower than in liver, 21 times lower than in the uterus,8.4 times lower that in the blood and 13 times lower than in plasma.Since an unknown proportion of total brain radioactivity is due tocontamination by blood radioactivity, the values shown in Table X 1 forbrain radioactivity are an overestimate of the level of ¹⁴C(EM-800)—related radioactivity in the brain tissue itself. Such datasuggest that the level of the antiestrogen in the brain tissue is toolow, to counteract the effect of exogenous estrogen. It is important tonote that some of the radioactivity detected in the brain tissue may bedue to residual blood in the tissue (Table 14). Additionally, theradiochemical purity of the ¹⁴C-EM-800 used for this study was minimally96.25%.

TABLE 13 Mean Concentration of Drug-Derived Radioactivity (ng EM-800equiv/g tissue) in Selected Tissues of Female Long-Evans Rats Followinga Single Oral Dose of ¹⁴C-EM-800 (20 mg/kg)^(a) Time Brain Blood Plasma(hr) Mean^(b) (% CV) Mean^(b) (% CV) Mean^(b) (% CV) 2 17.6 (29) 148.7(22) 224.6 (20) 4 17.1 (29) 66.9 (45) 103.2 (39) 6 15.6 (8) 48.3 (29)74.1 (31) 8 16.8 (31) 41.1 (12) 64.1 (14) 12 10.0^(c) (87) 28.7 (54)40.7 (55) 24 0 (NC) 4.7^(d) (173) 10.1 (86) 36 0 (NC) 0 (NC) 0 (NC) 48 0(NC) 0 (NC) 0 (NC) 72 0 (NC) 0 (NC) 0 (NC) 96 0 (NC) 0 (NC) 0 (NC) 168 0(NC) 0 (NC) 0 (NC) ^(a)Values from report tables for LREM 1129 (EM-800:Tissue Distribution and Excretion of Radioactivity Following a SingleOral Dose of ¹⁴C-EM-800 (20 mg/2 mL/kg) to Male and Female Long-EvansRats). ^(b)Limit of quantification (LOQ) of 1.2 ng EM-800 equivalent.^(c)One sample below the LOQ; 0 used in calculation of mean. ^(d)Twosamples below the LOQ; 0 used in calculation of mean. % CV: Coefficientof variation expressed as a percent, where n = 3. NC: Not calculated.

TABLE 14 Mean Concentration of Drug-Derived Radioactivity (μg EM-800equiv/g tissue) in Selected Tissues of Female Long-Evans Rats Followinga Single Oral Dose of ¹⁴C-EM-800 (20 mg/kg)^(a) Time Brain Liver UterusBlood Plasma (hr) Mean^(b) (% CV) Mean^(b) (% CV) Mean^(b) (% CV)Mean^(b) (% CV) Mean^(b) (% CV) 2 0.0176 (29) 7.2547 (30) 0.3675 (36)0.1487 (22) 0.2246 (20) 4 0.0171 (29) 3.2201 (48) 0.2866 (83) 0.0669(45) 0.1032 (39) 6 0.0156 (8) 2.7462 (8) 0.2757 (19) 0.0483 (29) 0.0741(31) 8 0.0168 (31) 2.7748 (8) 0.3332 (46) 0.0411 (12) 0.0641 (14) 120.0100^(c) (87) 1.8232 (38) 0.2407 (25) 0.0287 (54) 0.0407 (55) 24 0(NC) 0.6391 (52) 0.0837 (54) 0.0047^(d) (173) 0.0101 (86) 36 0 (NC)0.4034 (22) 0.0261 (15) 0 (NC) 0 (NC) 48 0 (NC) 0.2196 (37) 0.0238 (44)0 (NC) 0 (NC) 72 0 (NC) 0.1326 (4) 0 (NC) 0 (NC) 0 (NC) 96 0 (NC) 0.0944(15) 0 (NC) 0 (NC) 0 (NC) 168 0 (NC) 0.0348 (14) 0 (NC) 0 (NC) 0 (NC)^(a)Values from report tables for LREM 1129 (EM-800: Tissue Distributionand Excretion of Radioactivity Following a Single Oral Dose of¹⁴C-EM-800 (20 mg/2 mL/kg) to Male and Female Long-Evans Rats). B: Limitof quantification (LOQ) of 1.2 ng EM-800 equivalent. C: One sample belowthe LOQ; 0 used in calculation of mean. D: Two samples below the LOQ; 0used in calculation of mean. % CV: Coefficient of variation expressed asa percent, where n = 3. NC: Not calculated.

Example 10 Clinical Trial ERC-205

Étude De Phase II-III Randomisée Avec Contrôle Placebo Pour Évaluer LesEffets de la DHEA Sur Les Symptômes Vasomoteurs (Bouffées DeChaleur)—Phase II-III Placebo-Controlled, Study to Evaluate the Effectsof Dhea on Vasomotor Symptoms (Hot Flushes) in Postmenopausal Women.

Study Design Summary

As illustrated in FIG. 24, this was a randomized, placebo controlled,study to evaluate the effect of DHEA on reducing vasomotor symptoms (hotflushes) compared to placebo administration. Postmenopausal womenexperiencing moderate or severe hot flushes per week (as determined by atwo week diary) were randomized to receive a daily dose of eitherplacebo or 50 mg DHEA. Fifty evaluable participants (25 patients perarm) were treated for four months with a daily assessment of hot flushesrecorded in a diary completed by each participant.

Postmenopausal women aged 40 to 70 years with moderate or severe hotflushes per week, as confirmed by a two-week screening hot flush diarywere enrolled after signing informed consent. The protocol was approvedby the Institutional Review Board (IRB) of Le Centre Hospitalier del'Université Laval and by Health Canada.

Women had to satisfy either a or b or c:

-   -   a. No menses for at least one year, or;    -   b. FSH levels ≥40 mIU/mL (within 60 days prior to Day 1) in        women with no menses ≥6 months but <12 months, or        hysterectomized women who were premenopausal at the time of        hysterectomy, or;    -   c. Previous bilateral oophorectomy.

A normal PAP smear (which includes inflammatory changes) and a normalbilateral mammogram within 12 months of randomization had to beavailable.

An endometrial thickness of 4 mm or less at transvaginal ultrasonographywas required.

The primary endpoint was the change from Baseline in the weeklyfrequency of moderate to severe hot flushes at Week 16, after fourmonths of treatment. The objectives also included the change fromBaseline in the weekly frequency of all hot flushes and the change fromBaseline in the weekly weighted severity score.

The secondary endpoints were the safety evaluation of DHEA as well asquality of life.

The response endpoint is the patient's paper diary which was filled indaily to specify the number and type of hot flushes as follows:

0 None. 1 Mild = sensation of heat without perspiration. 2 Moderate =sensation of heat with perspiration and no cessation of activitynecessary. 3 Severe = sensation of heat with perspiration necessitatingcessation of activity. This includes night sweats.

The hot flush diary began as a Screening diary for two weeks prior torandomization whereby patients had to complete the diary daily,recording the number and severity of hot flushes. The patients had torecord an average of 50 or more moderate or severe hot flushes per weekover the two-week period to be eligible (i.e., at least 100 hot flushesdocumented on the two week Screening diary).

Once randomized, the patient completed eight, two-week hot flush diariesupon beginning study medication. The diaries had to be filled out on adaily basis. The first diary was completed over the first two weeks andbe returned on the two week visit. The second two-week diary wascompleted over the next two weeks of the first four week treatmentperiod and was returned at the four week visit. At 4, 8, 12 and 16 weekvisits, two two-week diaries for hot flushes were collected.

Diary and blinded medication began on the same day (ie, on day 1. Thepatient began recording hot flushes when she woke up on the same day sheplanned to begin taking the study medication).

Results

As illustrated in FIG. 25 and Table 15, the number of moderate to severehot flushes decreased from 70.7±4.5 per week at screening to 50.1±5.7 atweek 4 (N.S. US placebo), 40.2±6.1 at week 8, 34.7±5.8 at week 12(p<0.05 vs placebo) and 32.2±5.8 at week 16 (p<0.0.5 vs placebo).Placebo cause a 32.9% decrease compared to 54.5% for DHEA.

A similar effect was observed on the frequency of all hot flushes (FIG.26, Table 16) with a prescreening value of 75.5±4.4 hot flushes per weekto 55.3±5.8 at week 4 (N.S. vs placebo), 44.7±6.3 at week 8, 39.5±5.9 atweek 12 (p<0.05 vs placebo) and 36.0±5.7 at week 16 (p<0.05 vs placebo).Placebo caused a 34.9% decrease compared to 52.4% for DHEA.

When the hot flushes were attributed a score of 1 for mild, 2 formoderate and 3 for severe, it can be seen that the values went form187.1±13.9 to 87.2±15.8 in the DHEA groups compared to 196.3±13.6 to130±14.1 in the placebo groups at 16 weeks (p<0.05). Placebo caused a18.0% decrease versus 53.4% for DHEA, thus indicating a 3.0-fold higherefficacy of DHEA.

As illustrated in Table 18, the effect of DHEA was exerted at a greaterdegree on the moderate to severe hot flushes an effect betterillustrated on the weighted severity score when the value was reduced by99.1±15.6 with DHEA and 68.6±15.6 for placebo. This effect is betterillustrated in Table 19 where the mean number of hot flushes in thegroups of women having 71 or more hot flushes per week at screening wentfrom 94.7±7.9 to 57.8±8.3 in the placebo group went from 88.5±7.1 to31.6±11.6 in the group of women who received DHEA. Such data show a 65%(64.3% for DHEA versus 39.0% for placebo) greater inhibition by DHEA inthe women most affected by vasomotor symptoms. In fact, in women havingbetween 50 and 70 moderate to severe hot flushes per week, the numberwent from 56.7±1.3 per week at prescreening to 32.7±6.2 at week 6 (43.3%decrease) in women who received DHEA compared to a 24.7% decrease withplacebo, thus indicating a 43% inhibition by DHEA over the placeboeffect.

Since the number of mild hot flushes is relatively low (comparison ofTables 18 and 19), similar conclusions are found in Table 20. The numberof hot flushes in women having more than 70 mild, moderate plus severehot flushes is decreased from 91.0±7.0 per week at prescreening to36.3±11.5 at week 16 in women receiving DHEA (60% decrease). In womenreceiving placebo the number of all hot flushes goes from 100.4±7.9 atscreening to 61.3±8.9 at week 16 for a 39.9% decrease. Such data show a50% greater efficacy of DHEA in the women having the largest number ofhot flushes of all degrees of severity.

Analogous conclusions are reached when a weighted severity score is usedfor calculations (Table 21). When women having more than 70 hot flushesper week are considered, the score goes from 241.6±21.1 at screening to95.2±34.3 at week 16 in women receiving DHEA (61.6% decrease) while forplacebo, the value decreases from 242.0±21.6 to 141.3±20.1 at week 16(41.6% decrease). In women having between 50 and 70 hot flushes per weekat screening, the values go from 144.3±6.7 at screening to 81.6±13.6 atweek 16 in women who received DHEA (43.5% decrease) compared to values154.2±3.7 and 118.8±20.2 for the placebo groups (33.0% decrease).

Conclusion

The present data demonstrated the efficacy of 50 mg DHEA treatment foralleviating vasomotor symptoms as assessed by the significant decreasein the total number of moderate to severe hot flushes or all hotflushes, as well as by the significant reduction of the hot flush weeklyseverity weighted score.

TABLE 15 NUMBER OF MODERATE TO SEVERE HOT FLUSHES PER WEEK ScreeningGROUP VALUE (mean of 2 w) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week7 Week 8 PLACEBO n 25 25 25 25 25 25 25 24 24 MEAN 76.0 61.0 55.0 57.458.5 58.8 54.9 55.4 56.8 SEM 5.3 5.6 6.1 5.7 6.0 5.6 5.4 5.6 6.1 MIN52.5 5 3 3 4 3 1 0 0 MAX 16 8 127 116 110 125 111 117 104 118 DHEA n 2525 25 25 25 22 22 22 22 (50 mg) MEAN 70.7 58.6 51.0 49.1 50.1 44.4 43.039.6 40.2 SEM 4.5 4.8 4.8 5.3 5.7 6.2 6.2 6.5 6.1 MIN 50.5 26 5 2 7 0 00 0 MAX 145.5 129 121 110 131 121 114 123 103 NUMBER OF MODERATE TOSEVERE HOT FLUSHES PER WEEK GROUP VALUE Week 9 Week 10 Week 11 Week 12Week 13 Week 14 Week 15 Week 16 PLACEBO n 24 24 24 24 24 24 24 24 MEAN56.3 54.8 51.7 52.8 49.8 52.3 52.6 51.0 SEM 5.8 5.7 5.7 6.0 5.6 5.9 5.95.8 MIN 0 0 0 0 0 0 0 1 MAX 116 108 106 115 110 112 98 103 DHEA n 22 2222 22 22 22 22 22 (50 mg) MEAN 39.6 38.6 36.0 34.7 33.2 33.3 34.0 32.2SEM 5.7 5.6 5.6 5.8 5.6 6.0 6.0 5.8 MIN 0 0 0 0 0 0 0 0 MAX 107 110 107105 101 102 110 104 CM110108-7

TABLE 16 NUMBER OF ALL HOT FLUSHES PER WEEK Screening GROUP VALUE (meanof 2 w) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 PLACEBOn 25 25 25 25 25 25 25 24 24 MEAN 83.4 68.2 60.2 62.3 63.2 63.2 59.460.5 61.7 SEM 5.1 5.7 6.3 5.8 5.8 5.5 5.2 5.4 5.9 MIN 52.5 12 7 5 17 7 61 1 MAX 168 128 117 114 130 111 118 110 121 DHEA n 25 25 25 25 25 22 2222 22 (50 mg) MEAN 75.7 64.2 56.4 54.6 55.3 49.8 47.9 44.5 44.7 SEM 4.45.1 5.0 5.4 5.8 6.4 6.4 6.8 6.3 MIN 51 26 7 2 7 0 0 0 0 MAX 147 129 121110 131 121 114 123 103 NUMBER OF ALL HOT FLUSHES PER WEEK GROUP VALUEWeek 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 PLACEBO n24 24 24 24 24 24 24 24 MEAN 61.3 59.0 55.4 56.7 54.8 55.9 56.2 54.3 SEM5.7 5.7 5.9 6.1 5.6 6.0 6.0 5.8 MIN 0 0 0 0 0 3 6 8 MAX 118 109 110 121110 113 107 103 DHEA n 22 22 22 22 22 22 22 22 (50 mg) MEAN 44.3 43.140.9 39.5 37.5 37.6 37.5 36.0 SEM 5.9 5.8 5.7 5.9 5.5 5.9 5.9 5.7 MIN 00 0 0 0 0 0 0 MAX 107 110 108 105 101 102 110 104 CM110108-8

TABLE 17 WEIGHTED SEVERITY SCORE OF HOT FLUSHES PER WEEK Screening GROUPVALUE (mean of 2 w) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7Week 8 PLACEBO n 25 25 25 25 25 25 25 24 24 MEAN 196.3 158.6 140.8 147.5148.6 148.0 139.7 142.6 145.9 SEM 13.6 13.9 14.8 13.7 13.9 12.8 12.613.1 14.4 MIN 139.5 20 13 14 30 13 8 1 1 MAX 433.5 325 295 279 297 263275 253 278 DHEA n 25 25 25 25 25 22 22 22 22 (50 mg) MEAN 187.1 156.0137.5 132.5 135.7 120.9 115.2 108.3 109.4 SEM 13.9 13.6 13.6 14.4 16.116.8 17.3 18.6 17.1 MIN 116 66 15 4 20 0 0 0 0 MAX 404 353 353 326 386330 335 357 302 WEIGHTED SEVERITY SCORE OF HOT FLUSHES PER WEEK GROUPVALUE Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16PLACEBO n 24 24 24 24 24 24 24 24 MEAN 143.7 139.7 131.0 133.6 127.1132.7 133.6 130.0 SEM 13.7 13.4 13.8 14.4 13.4 14.4 14.2 14.1 MIN 0 0 00 0 3 6 10 MAX 270 253 251 273 257 262 232 242 DHEA n 22 22 22 22 22 2222 22 (50 mg) MEAN 106.5 104.5 98.2 92.7 90.1 90.9 91.8 87.2 SEM 15.715.7 15.5 15.2 15.0 15.8 16.4 15.8 MIN 0 0 0 0 0 0 0 0 MAX 314 327 322315 301 306 330 311 CM110108-9

TABLE 18 MEAN CHANGE IN FREQUENCY AND SEVERITY OF HOT FLUSHES (HF) FROMBASELINE Frequency of moderate-severe HF Frequency of all HF Weightedseverity score of HF GROUP VALUE Week 4 Week 8 Week 12 Week 16 Week 4Week 8 Week 12 Week 16 Week 4 Week 8 Week 12 Week 16 PLACE- n 25 24 2424 25 24 24 24 25 24 24 24 BO MEAN −17.4 −19.8 −23.8 −25.7 −20.1 −22.1−27.1 −29.5 −47.7 −52.7 −65.0 −68.6 SEM 5.6 6.0 5.8 5.7 5.2 5.6 6.0 5.714.1 15.8 15.7 15.6 MIN −70.5 −78.5 −81.5 −80.5 −66 −77.5 −85.5 −87.5−187.5 −206.5 −227.5 −228.5 MAX 21 18 30 21 15.5 9.5 24.5 15.5 51.5 6162 71 DHEA n 25 22 22 22 25 22 22 22 25 22 22 22 (50 mg) MEAN −20.6−30.1 −35.6 −38.1 −20.4 −31.0 −36.2 −39.8 −51.4 −76.9 −93.6 −99.1 SEM5.3 6.2 6.5 5.9 5.3 6.1 6.5 5.9 14.0 16.6 17.4 15.6 MIN −74 −84 −82−76.5 −74.5 −75 −82 −77 −188.5 −202 −240.5 −205 MAX 27 18.5 29.5 25.525.5 18.5 29 25 77.5 39 57.5 53.5 CM110108-10

TABLE 19 NUMBER OF MODERATE TO SEVERE HOT FLUSHES PER WEEK ScreeningGROUP STRATA VALUE (mean of 2 w) Week 1 Week 2 Week 3 Week 4 Week 5 Week6 Week 7 Week 8 PLACEBO 50 to 70 n 13 13 13 13 13 13 13 12 12 hotflushes MEAN 58.7 46.8 40.8 49.5 48.2 48.6 47.1 46.3 48.0 per week SEM1.4 5.8 7.2 7.6 6.4 6.3 6.1 6.2 6.9 at screening MIN 52.5 5 3 3 4 3 1 00 MAX 66 81 88 101 87 88 88 73 80 ≥71 n 12 12 12 12 12 12 12 12 12 hotflushes MEAN 94.7 76.3 70.3 66.1 69.8 69.8 63.3 64.5 65.7 per week SEM7.9 7.9 8.0 8.1 9.6 8.5 8.8 8.9 9.7 at screening MIN 71 21 22 18 18 2013 9 10 MAX 168 127 116 110 125 111 117 104 118 DHEA 50 to 70 n 14 14 1414 14 13 13 13 13 (50 mg) hot flushes MEAN 56.7 54.2 49.0 46.5 44.0 38.339.8 34.7 38.1 per week SEM 1.3 4.7 4.9 6.2 5.7 6.2 6.4 5.6 6.2 atscreening MIN 50.5 26 5 2 7 0 0 0 0 MAX 70.5 90 81 90 82 82 72 70 75 ≥71n 11 11 11 11 11 9 9 9 9 hot flushes MEAN 88.5 64.1 53.5 52.5 57.8 53.247.6 46.7 43.3 per week SEM 7.1 9.0 9.3 9.2 10.7 12.0 12.5 13.9 12.5 atscreening MIN 72.5 26 22 15 10 4 5 6 0 MAX 145.5 129 121 110 131 121 114123 103 NUMBER OF MODERATE TO SEVERE HOT FLUSHES PER WEEK GROUP STRATAVALUE Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16PLACEBO 50 to 70 n 12 12 12 12 12 12 12 12 hot flushes MEAN 50.8 49.244.1 47.9 44.9 46.3 46.8 44.2 per week SEM 7.0 7.1 6.7 7.7 6.9 8.7 8.57.9 at screening MIN 0 0 0 0 0 0 0 1 MAX 90 86 83 96 77 100 98 87 ≥71 n12 12 12 12 12 12 12 12 hot flushes MEAN 61.8 60.4 59.3 57.8 54.7 58.258.4 57.8 per week SEM 9.2 8.8 9.0 9.4 8.8 8.1 8.1 8.3 at screening MIN7 6 2 7 9 10 8 8 MAX 116 108 106 115 110 112 98 103 DHEA 50 to 70 n 1313 13 13 13 13 13 13 (50 mg) hot flushes MEAN 38.5 36.6 35.6 35.8 32.834.2 33.7 32.7 per week SEM 6.2 6.4 6.2 6.8 6.6 7.2 6.6 6.2 at screeningMIN 0 0 0 0 0 0 0 0 MAX 73 75 70 80 78 77 74 76 ≥71 n 9 9 9 9 9 9 9 9hot flushes MEAN 41.1 41.6 36.4 33.1 33.9 32.0 34.4 31.6 per week SEM11.2 10.6 10.9 10.5 10.4 10.8 11.7 11.6 at screening MIN 6 12 6 1 3 4 51 MAX 107 110 107 105 101 102 110 104 CM110108-11

TABLE 20 NUMBER OF ALL HOT FLUSHES PER WEEK Screening GROUP STRATA VALUE(mean of 2 w) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8PLACEBO 50 to 70 n 13 13 13 13 13 13 13 12 12 hot flushes MEAN 67.7 54.345.4 53.8 53.2 52.8 51.5 50.9 53.3 per week SEM 2.2 5.2 7.5 7.6 5.8 6.05.5 5.4 6.3 at screening MIN 52.5 12 7 5 17 7 6 1 1 MAX 78.5 83 92 10287 91 88 73 81 ≥71 n 12 12 12 12 12 12 12 12 12 hot flushes MEAN 100.483.3 76.2 71.5 74.2 74.4 68.0 70.2 70.1 per week SEM 7.9 8.6 8.3 8.3 9.68.4 8.5 8.7 9.7 at screening MIN 71 32 34 32 34 35 32 22 23 MAX 168 128117 114 130 111 118 110 121 DHEA 50 to 70 n 14 14 14 14 14 13 13 13 13(50 mg) hot flushes MEAN 63.7 60.8 55.3 52.3 49.4 42.7 43.7 39.2 41.8per week SEM 3.0 5.9 5.7 6.7 6.3 6.9 7.0 6.7 7.0 at screening MIN 51 307 2 7 0 0 0 0 MAX 82 106 84 90 87 82 72 72 86 ≥71 n 11 11 11 11 11 9 9 99 hot flushes MEAN 91.0 68.6 57.9 57.5 62.7 60.0 53.9 52.2 48.9 per weekSEM 7.0 9.1 9.1 9.2 10.4 11.6 12.3 13.7 12.0 at screening MIN 72.5 26 2415 22 14 13 7 9 MAX 147 129 121 110 131 121 114 123 103 NUMBER OF ALLHOT FLUSHES PER WEEK GROUP STRATA VALUE Week 9 Week 10 Week 11 Week 12Week 13 Week 14 Week 15 Week 16 PLACEBO 50 to 70 n 12 12 12 12 12 12 1212 hot flushes MEAN 55.4 53.8 48.1 51.4 49.1 49.8 49.8 47.3 per week SEM6.4 6.7 6.6 7.2 6.7 8.3 7.8 7.2 at screening MIN 0 0 0 0 0 3 6 8 MAX 9088 83 96 86 100 98 87 ≥71 n 12 12 12 12 12 12 12 12 hot flushes MEAN67.2 64.3 62.7 61.9 60.4 62.0 62.6 61.3 per week SEM 9.5 9.2 9.6 10.08.9 8.6 8.9 8.9 at screening MIN 16 11 4 11 12 14 13 9 MAX 118 109 110121 110 113 107 103 DHEA 50 to 70 n 13 13 13 13 13 13 13 13 (50 mg) hotflushes MEAN 42.4 40.5 39.5 39.7 36.2 37.8 36.5 35.7 per week SEM 7.06.8 6.6 7.1 6.6 7.3 6.5 5.8 at screening MIN 0 0 0 0 0 0 0 0 MAX 81 7570 80 78 77 74 76 ≥71 n 9 9 9 9 9 9 9 9 hot flushes MEAN 47.0 46.9 42.839.3 39.3 37.3 38.8 36.3 per week SEM 10.8 10.6 10.7 10.6 9.9 10.5 11.411.5 at screening MIN 14 12 10 2 5 4 5 1 MAX 107 110 108 105 101 102 110104 CM110108-12

TABLE 21 WEIGHTED SEVERITY SCORE OF HOT FLUSHES PER WEEK Screening GROUPSTRATA VALUE (mean of 2 w) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6Week 7 Week 8 PLACEBO 50 to 70 n 13 13 13 13 13 13 13 12 12 hot flushesMEAN 154.2 127.2 107.7 129.9 128.1 127.6 125.0 125.7 131.1 per week SEM3.7 14.3 17.6 18.2 14.8 15.0 14.4 15.0 17.1 at screening MIN 139.5 20 1314 30 13 8 1 1 MAX 178.5 228 206 242 210 211 207 184 214 ≥71 n 12 12 1212 12 12 12 12 12 hot flushes MEAN 242.0 192.8 176.6 166.6 170.9 170.0155.7 159.5 160.7 per week SEM 21.6 20.7 20.2 20.0 23.1 20.0 20.6 21.023.0 at screening MIN 164 63 67 62 63 65 53 48 44 MAX 433.5 325 295 279297 263 275 253 278 DHEA 50 to 70 n 14 14 14 14 14 13 13 13 13 (50 mg)hot flushes MEAN 144.3 141.0 127.1 118.9 111.6 97.8 99.0 88.3 95.8 perweek SEM 6.7 13.1 12.6 15.4 12.8 15.2 15.0 13.9 14.6 at screening MIN116 73 15 4 20 0 0 0 0 MAX 208.5 239 211 234 182 171 163 147 182 ≥71 n11 11 11 11 11 9 9 9 9 hot flushes MEAN 241.6 175.2 150.8 149.8 166.5154.1 138.7 137.2 129.0 per week SEM 21.1 25.8 26.7 26.3 31.2 32.7 36.140.2 36.4 at screening MIN 176 66 57 35 38 27 31 16 19 MAX 404 353 353326 386 330 335 357 302 WEIGHTED SEVERITY SCORE OF HOT FLUSHES PER WEEKGROUP STRATA VALUE Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week15 Week 16 PLACEBO 50 to 70 n 12 12 12 12 12 12 12 12 hot flushes MEAN136.8 131.3 118.0 127.5 119.3 122.4 124.0 118.8 per week SEM 16.9 16.516.5 18.9 17.2 21.6 20.7 20.2 at screening MIN 0 0 0 0 0 3 6 10 MAX 210203 196 219 196 234 219 224 ≥71 n 12 12 12 12 12 12 12 12 hot flushesMEAN 150.6 148.1 144.1 139.7 135.0 142.9 143.2 141.3 per week SEM 22.121.5 22.1 22.5 21.0 19.5 20.0 20.1 at screening MIN 29 22 8 23 26 30 2622 MAX 270 253 251 273 257 262 232 242 DHEA 50 to 70 n 13 13 13 13 13 1313 13 (50 mg) hot flushes MEAN 96.0 92.7 89.8 88.5 81.4 87.0 83.7 81.6per week SEM 14.8 15.1 14.4 15.5 14.8 16.5 14.8 13.6 at screening MIN 00 0 0 0 0 0 0 MAX 174 156 169 193 190 192 180 189 ≥71 n 9 9 9 9 9 9 9 9hot flushes MEAN 121.8 121.6 110.2 98.7 102.8 96.6 103.6 95.2 per weekSEM 32.6 32.1 32.6 31.0 30.6 31.5 34.9 34.4 at screening MIN 33 31 22 49 10 11 3 MAX 314 327 322 315 301 306 330 311 CM110108-13

Example 11 Clinical Trial ERC-213

DHEA Bioavailability Following Administration of Vaginal Suppositoriesin Post-Menopausal Women with Vaginal Atrophy

Study Design Summary

The primary objective of that study was measurement of the maturationvalue of the vaginal epithelial cells following daily intravaginalapplication of DHEA. Forty postmenopausal women were randomized toreceive a daily dose of one ovule of the following DHEA concentrations:0.0%, 0.5% (6.5 mg of DHEA/ovule), 1.0% (13 mg of DHEA/ovule) or 1.8%(23.4 mg of DHEA/ovule) for 7 days. The systemic bioavailability of DHEAand its metabolites were also measured.

Results

After only one week of daily administration of the DHEA suppositories,the maturation index increased by 107% (p<0.01), 75% (p<0.05) and 150%(p<0.01) in the 0.5%, 1.0% and 1.8% DHEA groups, respectively (FIG. 27).No change was observed in the placebo group between day 1 and day 7.Vaginal pH, on the other hand, decreased from 6.29±0.21 to 5.75±0.27(p<0.05), 6.47±0.23 to 5.76±0.22 (p<0.01) and 6.53±0.25 to 5.86±0.28(p<0.05), respectively in the 0.5%, 1.0% and 1.8% DHEA groups (FIG. 27).No change of vaginal pH was observed in the placebo group.

Conclusion

The present data show that the intravaginal administration of DHEApermits to rapidly achieve the beneficial effects against vaginalatrophy without significant changes of serum estrogens, thus avoidingthe increased risk of breast cancer associated with the currentintravaginal or systemic estrogenic formulations and adding the localbenefits on all the layers of the vagina of the recently recognizedandrogenic component of DHEA action in this tissue.

PHARMACEUTICAL COMPOSITION EXAMPLES

Set forth below, by way of example and not of limitation, are severalpharmaceutical compositions utilizing preferred active SERM Acolbifene(EM-652.HCl; EM-1538) and preferred active sex steroid precursordehydroepiandrosterone (DHEA, Prasterone). Other compounds of theinvention or combination thereof, may be used in place of (or inaddition to) Acolbifene or dehydroepiandrosterone. The concentration ofactive ingredient may be varied over a wide range as discussed herein.The amounts and types of other ingredients that may be included are wellknown in the art.

Example A Pharmaceutical Composition for Orally Administration(Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA10.0 Lactose hydrous 70.0 Starch 4.8 Cellulose microcrystalline 9.8Magnesium stearate 0.4

Example B Pharmaceutical Composition for Orally Administration (Tablets)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Gelatin 5.0 Lactose 58.5 Starch 16.5

Example C Topical Administration (Cream)

Weight % Ingredient (by weight of total composition) DHEA 1.0 Acolbifene0.2 Emulsifying Wax, NF 18.0 Light mineral oil, NF 12.0 Benzyl alcohol1.0 Ethanol 95% USP 33.8 Purifed water, USP 34.0

Example D Vaginal Administration Vaginal Suppository or Ovule

Weight % Ingredient (by weight of total composition) DHEA 0.25 to 2.0Acolbifene 0.25 to 3.0 Witepsol H-15 base  95.0 to 99.5

DHEA suppositories were prepared using Witepsol H-15 base (Medisca,Montreal, Canada). Any other lipophilic base such as Hard Fat,Fattibase, Wecobee, cocoa butter, theobroma oil or other combinations ofWitepsol bases could used. Preferred SERMs are EM-800, and Acolbifene

KIT EXAMPLES

Set forth below, by way of example and not of limitation, are severalkits utilizing preferred active SREM Acolbifene, preferred antiestrogenFaslodex and preferred active a sex steroid precursor DHEA. Theconcentration of active ingredient may be varied over a wide range asdiscussed herein. The amounts and types of other ingredients that may beincluded are well known in the art.

Example D Kit The SERM and Sex Steroid Precursor are Orally AdministeredNon-Steroidal Antiestrogen Composition for Oral Administration(Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4+

DHEA Composition for Oral Administration (Gelatin Capsule)

Weight % Ingredient (by weight of total composition) DHEA 25.0 Lactosehydrous 27.2 Sodium Starch Glycolate 20.0 Microcrystalline Cellulose,Colloidal 27.2 Silicon Dioxide, Silica Colloidal Anhydrous and LightAnhydrous Silicic Acid Colloidal Silicon Dioxide 0.1 Magnesium stearate0.5

Other SERMs may be substituted for Acolbifene in the above formulations,as well as other sex steroid precursors may be substituted for DHEA.More than one SERM or more than one sex steroid precursor may beincluded in which case the combined weight percentage is preferably thatof the weight percentage for the single sex steroid precursor or singleSERM given in the examples above.

Example E Kit The SERM is Orally Administered and the Sex SteroidPrecursor is Intra Vaginally Administered SERM Composition For OralAdministration (Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4+

Vaginal Suppository

Weight % Ingredient (by weight of total composition) DHEA 0.25 to 2.0   Witepsol H-15 base 98 to 99.75

DHEA suppositories were prepared using Witepsol H-15 base (Medisca,Montreal, Canada). Any other lipophilic base such as Hard Fat,Fattibase, Wecobee, cocoa butter, theobroma oil or other combinations ofWitepsol bases could used.

Example F Kit The SERM and the Sex Steroid Precursor are Intra VaginallyAdministered Vaginal Suppository

Weight % Ingredient (by weight of total composition) DHEA 0.25 to 2.0   Witepsol H-15 base 98 to 99.75+

Vaginal Suppository

Weight % Ingredient (by weight of total composition) Acolbifene 0.3 to3.0 Hard Fat 97.0 to 99.7

Acolbifene suppositories were prepared using Hard Fat (Witepsol). Anyother bases such as Fattibase, Wecobee, cocoa butter, theobroma oil orother combinations of Hard Fat could be used.

Example G The SERM is Orally Administered and the Sex Steroid Precursoris Percutaneously Administered SERM Composition for Oral Administration(Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4+

Sex Steroid Precursor Composition For Oral Administration (Gel)

Weight % Ingredient (by weight of total composition) DHEA 2.0Caprylic-capric Triglyceride (Neobee 5.0 M-5) Hexylene Glycol 15.0Transcutol (diethyleneglycol 5.0 monomethyl ether) Benzyl alcohol 2.0Cyclomethicone (Dow corning 345) 5.0 Ethanol (absolute) 64.0Hydroxypropylcellulose (1500 cps) 2.0 (KLUCEL)or

Sex Steroid Precursor Composition For Oral Administration (Cream)

Weight % (by weight of total composition) Ingredient FormulationEM-760-48-1.0% Cyclometicone 5.0% Light mineral oil 3.0% 2-ethylhexylstearate 10.0% Cutina E24 1.0% DC emulsifier 10 3.0% BHT 0.09%Propyleneglycol 46.01% Ethanol 95 10.0% DHEA 1.0% Eau purifiée 15.0%MgSO4 0.65% Ethanol 95 5.25% Total 100.0%

Example H Kit The Antiestrogen is Intramuscularly Administered and SexSteroid Precursor is Orally Administered Commercially AvailableSteroidal Antiestrogen Faslodex

+

DHEA Composition for Oral Administration (Gelatin Capsule)

Weight % Ingredient (by weight of total composition) DHEA 25.0 Lactosehydrous 27.2 Sodium Starch Glycolate 20.0 Microcrystalline Cellulose,Colloidal 27.2 Silicon Dioxide, Silica Colloidal Anhydrous and LightAnhydrous Silicic Acid Colloidal Silicon Dioxide 0.1 Magnesium stearate0.5

Other SERMs (Toremifene, Ospemifene, Raloxifene, Arzoxifene,Lasofoxifene, TSE-424, ERA-923, EM-800, SERM 3339, GW-5638) may besubstituted for Acolbifene in the above formulations, as well as othersex steroid inhibitors may be substituted for DHEA. More than one SERMor more than one precursor may be included in which case the combinedweight percentage is preferably that of the weight percentage for thesingle precursor or single SERM given in the examples above.

The invention has been described in terms of preferred embodiments andexamples, but is not limited thereby. Those of skill in the art willreadily recognize the broader applicability and scope of the inventionwhich is limited only by the patent claims herein.

What is claimed is:
 1. A method of preventing and treating loss ofcognition, said method comprising administering to a postmenopausalwoman in need of said treatment, (i) an amount of a sex steroidprecursor selected from the group consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol and4-androstene-3,17-dione, in combination with (ii) an amount of aselective estrogen receptor modulator, wherein the modulator is EM-652or a pharmaceutically acceptable salt thereof, and wherein said amountsare sufficient to achieve said prevention and treatment.
 2. The methodof claim 1, where said selective estrogen receptor modulator is:

and wherein the selective estrogen receptor modulator is an opticallyactive compound.
 3. The method of claim 1, wherein the selectiveestrogen receptor modulator is a EM-652 salt of an acid selected fromthe group consisting of acetic acid, adipic acid, benzenesulfonic acid,benzoic acid, camphorsulfonic acid, citric acid, fumaric acid,hydroiodic acid, hydrobromic acid, hydrochloric acid,hydrochlorothiazide acid, hydroxy-naphthoic acid, lactic acid, maleicacid, methanesulfonic acid, methylsulfuric acid,1,5-naphthalenedisulfonic acid, nitric acid, palmitic acid, pivalicacid, phosphoric acid, propionic acid, succinic acid, sulfuric acid,tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valericacid.
 4. The method of claim 1, wherein said selective estrogen receptormodulator is:

wherein the selective estrogen receptor modulator is an optically activecompound; and wherein the sex steroid precursor isdehydroepiandrosterone.
 5. The method of claim 1, wherein said selectiveestrogen receptor modulator is intravaginally administered.
 6. Themethod of claim 2, wherein the selective estrogen receptor modulator isintravaginally administered.
 7. The method of claim 1, wherein theselective estrogen receptor modulator is orally administered.
 8. Themethod of claim 1, wherein the selective estrogen receptor modulator ispercutaneously administered.
 9. The method of claim 1, wherein theamount of selective estrogen receptor modulator decreases the risk ofbreast, uterine and endometrial cancer normally occurring in saidpostmenopausal women and to prevent bone loss, osteoporosis,hypertension, insulin resistance, diabetes, obesity and atherosclerosis.10. The method of claim 1, wherein the loss of cognition is memory loss.